Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-18
2001-07-10
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007360, C435S023000, C435S032000, C435S034000, C435S091200, C536S023700
Reexamination Certificate
active
06258532
ABSTRACT:
BACKGROUND OF THE INVENTION
It has long been recognized that the appropriate use of susceptibility testing allows the most effective use of antimicrobial agents for the therapy of infectious diseases (1,2). Susceptibility testing for microorganisms such as the chlamydiae that cannot be cultured without the use of animal or tissue cultures is well recognized as being quite difficult (3,4). Early work used embryonated egg yolk sacs, and animal models, but these techniques were slow and cumbersome (5). In vitro susceptibility testing of chlamydiae is currently done using tissue culture cell lines (3-10). In these cell culture procedures, cycloheximide or a similar agent is routinely used to impair host cell metabolism and thus provide intracellular conditions in the host cell that enhance chlamydial growth. The use of cycloheximide has been found to increase the size and visibility of the chlamydial inclusion bodies. After a period of incubation, visual detection of inclusion bodies or immunochemical detection of chlamydial antigen is the endpoint (3,4). The minimal inhibitory concentration (MIC) is generally defined as the lowest concentration of antimicrobial agent at which no inclusion is seen after incubation. The minimal chlamydiacidal concentration (MCC) is defined as the lowest concentration of antimicrobial agent at which no inclusion is seen after several passages.
The murine model has been used extensively for the in vivo evaluation of chlamydial infection (11-18). Therefore, it is not surprising that in addition to in vitro cell culture methods, the murine model of chlamydial infection is also used for in vivo susceptibility testing (17).
Susceptibility testing of chlamydiae, including the most recent species
C. pneumoniae
, has been relatively extensive considering the difficulties encountered in testing an intracellular microorganism (3,5,10,19-21), and the results are considered to be consistent (5). However, in vitro susceptibility testing methods for chlamydiae are not standardized in terms of the testing conditions (3,4). Standardization of testing conditions for susceptibility testing is a well recognized requirement in general (1,2) and likewise should be required for chlamydiae (3). Moreover, results from in vitro susceptibility testing methods using current tissue culture conditions may not reflect the results seen with in vivo conditions (1-3). For example, Wyrick et al. (22) has shown that susceptibility testing results were different with polarized human endothelial cells as opposed to nonpolarized cells. Other conditions of testing have been found to markedly influence the results of chlamydial susceptibility testing (17). The timing of the addition of the antimicrobial agents to the cell culture is particularly important: the addition of agents before infection of the cell culture may lower the MICs and MCCs by 8-fold (23). Accordingly, the antimicrobial agents are usually added 30 to 60 minutes after the cells are infected (3,4).
Another common difficulty is determining the endpoint of the susceptibility test. This is a critical issue in both in vitro and in vivo methods (1-3). Visualization of the inclusion body by fluorescent microscopy is usually done (4) and is known to be observer-dependent (3). Attempts at achieving a more accurate and less subjective endpoint have been made. Kahn and colleagues (24) have recently reported a reverse transcriptase-PCR based assay for in vitro susceptibility testing of
Chlamydia pneumoniae
which avoids many of the problems associated with the determination of the endpoint. This method uses Southern hybridization to detect PCR-amplified messenger RNA. These investigators found that the use of this test method resulted in higher MICs and MCCs for a test strain as compared to results with conventional methods. It should be noted, however, that since messenger RNA is only present in active, metabolizing bacteria, this endpoint only indicates the presence or absence of active bacteria.
Another potential problem with current susceptiblity testing methods is the routine use of cycloheximide. The effect of antimicrobial agents against metabolizing chlamydiae requires that the agent penetrate the infected cell. The physiochemical properties of drugs are the main factors that influence their distribution in tissues and penetration in cells. However, the penetration of antimicrobial agents into the host cells can be greatly influenced by energy-requiring mechanisms such as active transport of the agent into the host cell or active efflux of the agent out of the host cell. The use of cycloheximide negates such mechanisms in the host cell. The measurement of antimicrobial levels in cells is a recognized problem and needs additional research (25). Until such work is done, the potential influence of cycloheximide on the penetration of antimcrobial agents is probably best avoided by selecting a different endpoint in which visualization of the inclusion body is not used.
The murine model for the therapy for chlamydial infections has been used more frequently in an attempt to avoid some of the problems encountered with in vitro susceptibility test methods. One group of investigators have demonstrated discrepancies between the in vitro MICs and survival rates (26,27). This data suggests that an in vivo animal model is more predictive of clinical outcome than is the current cell culture system. Unfortunately, the end point of such animal studies is still a problem. Although survival is a clearly discernible endpoint, it does not address the issue of cryptic infections which may be present in the survivors. If antimicrobial therapy can induce cryptic chlamydial infection, and cryptic infection then causes chronic diseases such as atherosclerosis, a method for detecting cryptic infection in animal models is needed.
SUMMARY OF THE INVENTION
The invention pertains to in vitro and in vivo susceptibility tests for identifying agent(s) capable of significantly reducing/eliminating infection caused by intracellular pathogens, such as Chlamydia and particularly
Chlamydia pneumoniae
. The methods comprise preparing tissue culture from cell lines; inoculating these cells with intracellular pathogen (e.g., Chlamydia) in the absence of cycloheximide; allowing the pathogen to infect these cells for several days; adding agent(s) to be tested, which agent(s) is/are replaced as needed for the duration of incubation; isolating pathogen nucleic acid from the cells; and assessing the presence or absence of pathogen DNA using a suitable nucleotide amplification assay, such as PCR. Preferably the presence or absence of signal for amplified DNA encoding a protein of the intracellular pathogen is determined. In one embodiment, the presence or absence of DNA encoding MOMP of Chlamydia or other chlamydial protein is determined. Absence of a signal indicates a reduction in the degree of infection below that which is detectable by nucleic acid amplification techniques and strongly suggests eradication of the microorganism. The susceptibility tests described herein are particularly useful as a drug screening tool for assessing the activity of single agents or combinations of agents against intracellular pathogen infection.
The invention is described below with regard to Chlamydia species and
Chlamydia pneumoniae
in particular as illustrative of the intracellular pathogen. However, it should be understood that other intracellular pathogens are embraced by this invention.
The unique and novel aspect of the susceptabilty test described here within is that it measures the presence or absence of chlamydial DNA and thus can detect cryptic forms and/or elementary bodies both of which are infectious, yet are not replicating.
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patent: 1 156 589 (1997-08
Mitchell William M.
Stratton Charles W.
Clark & Elbing LLP
Johannsen Diana
Myers Carla J.
Vanderbilt University
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