Two general types of in vitro tests are used to monitor antimicrobial therapy: measure¬ment of blood or body fluid antibiotic activity against the responsible organism, and assay of actual antibiotic concentrations in blood or other bodily fluids.
I. The serum bactericidal test determines the killing power” of patient serum against the infecting organism. The result is expressed as the highest dilution of serum that will produce the desired effect.
A. Method. The serum bactericidal test involves a modification of the broth dilution technique. Serum usually is obtained from the patient at times believed to correlate with the maximum or minimum antibacterial activity. Serial, twofold dilutions of the patient’s serum are inoculated with a standard quantity of the infecting organism. After overnight incubation of the mixtures, the inhibitory and lethal end points are determined as in broth dilution susceptibility testing, shown sche¬matically in Fig. 25-3. The serum inhibitory or bacteriostatic activity is defined as the highest dilution of serum that demonstrates a visible inhibitory effect. The serum lethal or bactericidal activity is similarly expressed as the highest dilution that produces a lethal effect, usually defined as a 99.9% or greater reduction of viable organisms in the initial inoculum. Bactericidal activity of other bodily fluids such as CSF, urine, and synovial fluid also can he measured by a modification of this method.
1. Variables. The measurement of serum bactericidal activity is influenced by numerous technical variables. These include the type of serum or broth diluents used, whether serum complement is or is not inactivated, the concentration of magnesium and calcium ions in the media, and the definition of the bacteri¬cidal end point. For example, a patient’s serum containing highly protein-bound antibiotics may show greater bactericidal activity if diluted in nutrient broth (which has a low protcin concentration) rather than in pooled human serum (which has a high protein concentration). The lack of interlaboratory standardization in the performance of these tests makes it difficult to com¬pare results between studies (43, 43a].
2. Timing of sample. Several authors favor collection of the serum sample at peak, whereas others prefer trough levels. The utility of peak versus trough serum bactericidal activity remains controversial [431.
B. Clinical application. Many authorities advocate using serum inhibitory or bacteri¬cidal activity as the best indicator of potential therapeutic efficacy. The test is the most reliable in vitro correlate of actual in vivo conditions because it accounts for other components of the antibacterial activity of serum in addition to the antibiotic (i.e., serum complement, opsonins, lysozymes). However, clinical appli¬cability of the serum bactericidal titer remains to be proven rigorously [431, and its use remains somewhat controversial [18, 44]. infectious disease consultation is advised to assist in the appropriate utilization and interpretation of serum bactericidal tests.
A determination of serum bactericidal activity may prove useful in guiding therapy, particularly in the following situations:
1. Endocarditis may be more effectively treated when higher serum bactericidal activity can be achieved. However, the results of serum bactericidal tests are not necessarily predictive of survival or clinical cure, and the peak and trough bactericidal titers that best correlate with outcome are not yet clear [43, 43a, 451. Although a peak bactericidal titer of at least 1:8 is most frequently recommended, one study concluded that a peak titer of 1:64 or more and a trough titer of 1:32 or more were most predictive of bacteriologic cure of endocarditis; the test was a poor predictor of bacteriologic failure [46].
The bactericidal titer may be particularly helpful in the following circum¬stances:
a. When endocarditis is caused by organisms that are not highly sensitive to the antibiotics being used, and a synergistic combination of antibiotics might be more effective
b. When less well established treatment regimens are employed
c. When the patient fails to improve on standard therapy
d. When the serum bactericidal titer is very high and drug toxicity is a significant risk, in which case the drug dose might be reduced without compromis¬ing antibacterial effect
2. In acute and chronic osteomyelitis, serum bactericidal titers that exceed cer¬tain levels have been correlated with cure [47]. When changing from parenteral to oral therapy of acute hematogenous osteomyelitis in children, bactericidal titers often are monitored to adjust antibiotic dosage to achieve a bactericidal level of 1:8 or more [43a]. The usefulness of serum bactericidal tests in the management of osteomyelitis, particularly in adults, remains uncertain.
3. In the immunocompromised host, a serum bactericidal titer of 1:8 or greater has been correlated with successful treatment of bacteremia and soft-tissue infections [48]. Higher bactericidal titers may be desirable in the granulocyto¬penic patient with gram-negative rod bacteremia [49].
4. In patients with acute pulmonary exacerbations of cystic fibrosis, peak serum bactericidal titers of 1:128 or greater against the patients’ pulmonary patho¬gens have been correlated with favorable bacteriologic responses to therapy [50].
II. Antimicrobial levels may be obtained to assess the adequacy of the chosen dose and route of administration and to avoid toxicity [19].
A. Methods
1. Correct timing of samples is necessary for accurate interpretation of the significance of antibiotic levels. The two measurements usually performed are the anticipated peak and trough blood levels of the antimicrobial after a dose has been given.
a. Peak blood levels usually are obtained 1 hour after an intramuscular dose, 30 minutes after the completion of an intravenous infusion, or 1—2 hours after an oral dose. In patients with renal insufficiency who receive antimicrobials by the parenteral route, peak levels may be delayed 2—4 hours after an intramuscular antibiotic dose or 1 hour after an intravenous dose.
b. Trough blood levels are obtained immediately before the next dose is due.
c. The blood should be obtained in tubes free of anticoagulant.
d. The sample should be taken promptly to the laboratory and quickly pro¬cessed. Some antibiotics rapidly lose activity, and the simultaneous pies¬ence of two antibiotics may result in one agent inactivating the other (e.g., carbenicillin can inactivate gentamicin).
e. The laboratory requisition should indicate clearly the antibiotic level de¬sired, time of most recent dose, amount of most recent dose, and any con¬comitant treatment with other antibiotics.
2. Techniques for assay of antibiotic levels. Prior to 1970, bioassays (agar diffu¬sion and broth dilution) were the most commonly used techniques to assay for levels of antibiotics in bodily fluids. l3ioassnys have been largely supplanted by a variety of more accurate and reproducible methods (e.g., immunoassays and high-pressure liquid chromatography),
a. Bioassays are performed by parallel dilution of both antibiotic standards and the patient’s bodily fluid. The dilutions then are tested for their ability to inhibit the growth of an indicator organism. The quantity of antibiotic in the bodily fluid is derived from the relationship between the degree of inhibition of the indicator organism by the bodily fluid and the inhibition by the antibiotic standards. Because bioassays depend on the inhibitory effects of an antibiotic on an organism, they lack specificity (i.e., they cannot differentiate between the effects of two or more antibiotics present in a bodily fluid). Therefore, it is essential to submit complete and accurate information about combination antimicrobial therapy with specimens sent for bioassay. With such information, the laboratory can sometimes circumvent the problem by technical manipulations (e.g., add beta-lacta¬mase to inhibit penicillias, use niultidrug-resistsnt indicator organisms, or remove antibiotics with cation-exchange resins). Most bioassay systems are not as precise as other types of assays but, when the tests are per¬formed carefully with adequate controls, the precision generally is adequate for clinical use.
b. Immunologic assays are presently the most widely used method for de¬termining antibiotic levels in bodily fluids. They exploit the specificity of the antigen-antibody (antimicrobial-antibody) reaction and use sophisticated instrumentation. More simple latex agglutination tests have also been developed and marketed for the semiquantitative assay of aminoglycoside antibiotics. Immunoassays have gained widespread acceptance because they are rapid, accurate, specific, and easier to perform than bioassays. Aminoglycoside and vancomycin levels now are routinely available in many laboratories using the immunoassay method.
c. High-pressure liquid chromatography is a method for separating com¬pounds; quantitation is subsequently achieved by analysis of the separated compounds. Liquid chromatographic procedures have been developed to measure almost all antibiotics in clinical specimens but are used most widely for chloramphenicol because no suitable immunoassay has been developed for this drug. Immunoassays generally are favored because they are simpler to perform.
B. Clinical application. Determination of antibiotic levels may be considered in the following situations:
1. When complicated or life-threatening infections exist secondary to organisms with MIC or MBC values near the maximum achievable levels of the antibiotic being used. Pneumonia and bacteremia due to gram-negative organisms may respond more favorably to treatment when therapeutic plasma levels of amino¬glycosides are achieved [51, 52]. A high peak concentration of aminoglycoside relative to the MIC for the infecting organism has been correlated with im¬proved clinical response to therapy [53J. For further discussion of the role of aminoglycoside levels, see Chap. 28H.
2. When one wishes to monitor therapy with an antibiotic that could have toxic side effects, particularly in the presence of altered hepatic or renal function (e.g., aminoglycosides; see Chap. 28H).
3. When an infection due to a sensitive organism is not responding to antibiotic treatment and all other therapeutic approaches have been optimized.
C. Interpretation. As a general guide, it is anticipated that an infection will respond to therapy if a level of antibiotic greater than the MIC of the infecting organism can be achieved at the site of infection. However, the relationship between achievable serum levels and response at an extravascular site of infection is variable. Also, factors other than an absolute serum level may be important (e.g., magnitude of level in comparison to MIC, duration of level above the MIC, and effect of serum protein binding). Determination of an antibiotic level is not a substitute for clinical judgment, and other therapeutic modalities must always be optimized (e.g., draining abscesses, removing foreign bodies, and bolstering host defense mechanisms).
I. The serum bactericidal test determines the killing power” of patient serum against the infecting organism. The result is expressed as the highest dilution of serum that will produce the desired effect.
A. Method. The serum bactericidal test involves a modification of the broth dilution technique. Serum usually is obtained from the patient at times believed to correlate with the maximum or minimum antibacterial activity. Serial, twofold dilutions of the patient’s serum are inoculated with a standard quantity of the infecting organism. After overnight incubation of the mixtures, the inhibitory and lethal end points are determined as in broth dilution susceptibility testing, shown sche¬matically in Fig. 25-3. The serum inhibitory or bacteriostatic activity is defined as the highest dilution of serum that demonstrates a visible inhibitory effect. The serum lethal or bactericidal activity is similarly expressed as the highest dilution that produces a lethal effect, usually defined as a 99.9% or greater reduction of viable organisms in the initial inoculum. Bactericidal activity of other bodily fluids such as CSF, urine, and synovial fluid also can he measured by a modification of this method.
1. Variables. The measurement of serum bactericidal activity is influenced by numerous technical variables. These include the type of serum or broth diluents used, whether serum complement is or is not inactivated, the concentration of magnesium and calcium ions in the media, and the definition of the bacteri¬cidal end point. For example, a patient’s serum containing highly protein-bound antibiotics may show greater bactericidal activity if diluted in nutrient broth (which has a low protcin concentration) rather than in pooled human serum (which has a high protein concentration). The lack of interlaboratory standardization in the performance of these tests makes it difficult to com¬pare results between studies (43, 43a].
2. Timing of sample. Several authors favor collection of the serum sample at peak, whereas others prefer trough levels. The utility of peak versus trough serum bactericidal activity remains controversial [431.
B. Clinical application. Many authorities advocate using serum inhibitory or bacteri¬cidal activity as the best indicator of potential therapeutic efficacy. The test is the most reliable in vitro correlate of actual in vivo conditions because it accounts for other components of the antibacterial activity of serum in addition to the antibiotic (i.e., serum complement, opsonins, lysozymes). However, clinical appli¬cability of the serum bactericidal titer remains to be proven rigorously [431, and its use remains somewhat controversial [18, 44]. infectious disease consultation is advised to assist in the appropriate utilization and interpretation of serum bactericidal tests.
A determination of serum bactericidal activity may prove useful in guiding therapy, particularly in the following situations:
1. Endocarditis may be more effectively treated when higher serum bactericidal activity can be achieved. However, the results of serum bactericidal tests are not necessarily predictive of survival or clinical cure, and the peak and trough bactericidal titers that best correlate with outcome are not yet clear [43, 43a, 451. Although a peak bactericidal titer of at least 1:8 is most frequently recommended, one study concluded that a peak titer of 1:64 or more and a trough titer of 1:32 or more were most predictive of bacteriologic cure of endocarditis; the test was a poor predictor of bacteriologic failure [46].
The bactericidal titer may be particularly helpful in the following circum¬stances:
a. When endocarditis is caused by organisms that are not highly sensitive to the antibiotics being used, and a synergistic combination of antibiotics might be more effective
b. When less well established treatment regimens are employed
c. When the patient fails to improve on standard therapy
d. When the serum bactericidal titer is very high and drug toxicity is a significant risk, in which case the drug dose might be reduced without compromis¬ing antibacterial effect
2. In acute and chronic osteomyelitis, serum bactericidal titers that exceed cer¬tain levels have been correlated with cure [47]. When changing from parenteral to oral therapy of acute hematogenous osteomyelitis in children, bactericidal titers often are monitored to adjust antibiotic dosage to achieve a bactericidal level of 1:8 or more [43a]. The usefulness of serum bactericidal tests in the management of osteomyelitis, particularly in adults, remains uncertain.
3. In the immunocompromised host, a serum bactericidal titer of 1:8 or greater has been correlated with successful treatment of bacteremia and soft-tissue infections [48]. Higher bactericidal titers may be desirable in the granulocyto¬penic patient with gram-negative rod bacteremia [49].
4. In patients with acute pulmonary exacerbations of cystic fibrosis, peak serum bactericidal titers of 1:128 or greater against the patients’ pulmonary patho¬gens have been correlated with favorable bacteriologic responses to therapy [50].
II. Antimicrobial levels may be obtained to assess the adequacy of the chosen dose and route of administration and to avoid toxicity [19].
A. Methods
1. Correct timing of samples is necessary for accurate interpretation of the significance of antibiotic levels. The two measurements usually performed are the anticipated peak and trough blood levels of the antimicrobial after a dose has been given.
a. Peak blood levels usually are obtained 1 hour after an intramuscular dose, 30 minutes after the completion of an intravenous infusion, or 1—2 hours after an oral dose. In patients with renal insufficiency who receive antimicrobials by the parenteral route, peak levels may be delayed 2—4 hours after an intramuscular antibiotic dose or 1 hour after an intravenous dose.
b. Trough blood levels are obtained immediately before the next dose is due.
c. The blood should be obtained in tubes free of anticoagulant.
d. The sample should be taken promptly to the laboratory and quickly pro¬cessed. Some antibiotics rapidly lose activity, and the simultaneous pies¬ence of two antibiotics may result in one agent inactivating the other (e.g., carbenicillin can inactivate gentamicin).
e. The laboratory requisition should indicate clearly the antibiotic level de¬sired, time of most recent dose, amount of most recent dose, and any con¬comitant treatment with other antibiotics.
2. Techniques for assay of antibiotic levels. Prior to 1970, bioassays (agar diffu¬sion and broth dilution) were the most commonly used techniques to assay for levels of antibiotics in bodily fluids. l3ioassnys have been largely supplanted by a variety of more accurate and reproducible methods (e.g., immunoassays and high-pressure liquid chromatography),
a. Bioassays are performed by parallel dilution of both antibiotic standards and the patient’s bodily fluid. The dilutions then are tested for their ability to inhibit the growth of an indicator organism. The quantity of antibiotic in the bodily fluid is derived from the relationship between the degree of inhibition of the indicator organism by the bodily fluid and the inhibition by the antibiotic standards. Because bioassays depend on the inhibitory effects of an antibiotic on an organism, they lack specificity (i.e., they cannot differentiate between the effects of two or more antibiotics present in a bodily fluid). Therefore, it is essential to submit complete and accurate information about combination antimicrobial therapy with specimens sent for bioassay. With such information, the laboratory can sometimes circumvent the problem by technical manipulations (e.g., add beta-lacta¬mase to inhibit penicillias, use niultidrug-resistsnt indicator organisms, or remove antibiotics with cation-exchange resins). Most bioassay systems are not as precise as other types of assays but, when the tests are per¬formed carefully with adequate controls, the precision generally is adequate for clinical use.
b. Immunologic assays are presently the most widely used method for de¬termining antibiotic levels in bodily fluids. They exploit the specificity of the antigen-antibody (antimicrobial-antibody) reaction and use sophisticated instrumentation. More simple latex agglutination tests have also been developed and marketed for the semiquantitative assay of aminoglycoside antibiotics. Immunoassays have gained widespread acceptance because they are rapid, accurate, specific, and easier to perform than bioassays. Aminoglycoside and vancomycin levels now are routinely available in many laboratories using the immunoassay method.
c. High-pressure liquid chromatography is a method for separating com¬pounds; quantitation is subsequently achieved by analysis of the separated compounds. Liquid chromatographic procedures have been developed to measure almost all antibiotics in clinical specimens but are used most widely for chloramphenicol because no suitable immunoassay has been developed for this drug. Immunoassays generally are favored because they are simpler to perform.
B. Clinical application. Determination of antibiotic levels may be considered in the following situations:
1. When complicated or life-threatening infections exist secondary to organisms with MIC or MBC values near the maximum achievable levels of the antibiotic being used. Pneumonia and bacteremia due to gram-negative organisms may respond more favorably to treatment when therapeutic plasma levels of amino¬glycosides are achieved [51, 52]. A high peak concentration of aminoglycoside relative to the MIC for the infecting organism has been correlated with im¬proved clinical response to therapy [53J. For further discussion of the role of aminoglycoside levels, see Chap. 28H.
2. When one wishes to monitor therapy with an antibiotic that could have toxic side effects, particularly in the presence of altered hepatic or renal function (e.g., aminoglycosides; see Chap. 28H).
3. When an infection due to a sensitive organism is not responding to antibiotic treatment and all other therapeutic approaches have been optimized.
C. Interpretation. As a general guide, it is anticipated that an infection will respond to therapy if a level of antibiotic greater than the MIC of the infecting organism can be achieved at the site of infection. However, the relationship between achievable serum levels and response at an extravascular site of infection is variable. Also, factors other than an absolute serum level may be important (e.g., magnitude of level in comparison to MIC, duration of level above the MIC, and effect of serum protein binding). Determination of an antibiotic level is not a substitute for clinical judgment, and other therapeutic modalities must always be optimized (e.g., draining abscesses, removing foreign bodies, and bolstering host defense mechanisms).
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