U.S. patent application number 09/025176 was filed with the patent office on 2001-05-31 for methods for in vitro susceptibility testing of chlamydia.
Invention is credited to MITCHELL, WILLIAM M, STRATTON, CHARLES W.
Application Number | 20010002421 09/025176 |
Document ID | / |
Family ID | 25430524 |
Filed Date | 2001-05-31 |
United States Patent
Application |
20010002421 |
Kind Code |
A1 |
STRATTON, CHARLES W ; et
al. |
May 31, 2001 |
METHODS FOR IN VITRO SUSCEPTIBILITY TESTING OF CHLAMYDIA
Abstract
Methods for determining the susceptibility of intracellular
pathogens, particularly Chlamydia, to single or combination of test
agents are described. The methods can be used for in vitro or in
vivo evaluation of agents that can be used as therapeutic agents in
the treatment/eradication of pathogen infection in general or to
target a specific infected organ. Assays which utilize nucleic
amplification techniques (e.g., PCR) to determine effectiveness of
the agent(s) evaluated are also described.
Inventors: |
STRATTON, CHARLES W;
(NASHVILLE, TN) ; MITCHELL, WILLIAM M; (NASHVILLE,
TN) |
Correspondence
Address: |
KAREN F. ELBING
CLARK AND ELBING
176 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
25430524 |
Appl. No.: |
09/025176 |
Filed: |
February 18, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09025176 |
Feb 18, 1998 |
|
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08911593 |
Aug 14, 1997 |
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Current U.S.
Class: |
536/23.1 ;
435/6.12; 435/6.15; 536/24.3; 536/24.32 |
Current CPC
Class: |
A61K 31/455 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101; A61K 31/455
20130101 |
Class at
Publication: |
536/23.1 ; 435/6;
536/24.3; 536/24.32 |
International
Class: |
C07H 021/04; C12Q
001/68; C07H 021/04 |
Claims
We claim:
1. A method for determining susceptibility of an intracellular
pathogen to test agent, comprising exposing intracelluar pathogen
in cell or animal culture to the test agent in the absence of
cyclohexamine, determining the presence or absence of the
intracellular pathogen by determining the presence or absence of a
gene for that intracellular pathogen in the culture.
2. The method of claim 1 wherein the test agent is a single agent
or plurality of different test agents.
3. The method of claim 1 wherein the intracellular pathogen is
Chlamydia.
4. The method of claim 3 wherein the Chlamydia is Chlamydia
pneumoniae.
5. The method of claim 4 wherein the gene for Chlamydia pneumoniae
is MOMP.
6. The method of claim 1 wherein the presence or absence of the
gene for the intracellular pathogen is determined by a nucleic acid
amplification technique.
7. The method of claim 6 wherein the nucleic acid amplification
technique is PCR.
8. A method for determining susceptibility to test agent of an
intracellular pathogen infection of a target organ in an infected
animal, comprising contacting the infected animal with test agent,
determining the presence or absence of the intracellular pathogen
in the organ by determining the presence or absence of a gene for
that intracellular pathogen in the organ.
9. The method of claim 8 wherein the test agent is a single agent
or plurality of different test agents.
10. The method of claim 8 wherein the intracellular pathogen is
Chlamydia.
11. The method of claim 10 wherein the Chlamydia is Chlamydia
pneumoniae.
12. The method of claim 11 wherein the gene for Chlamydia
pneumoniae is MOMP.
13. The method of claim 8 wherein the presence or absence of the
gene for the intracellular pathogen is determined by a nucleic acid
amplification technique.
14. The method of claim 13 wherein the nucleic acid amplification
technique is PCR.
15. The method of claim 8 wherein the organ is selected from the
group consisting of lung, liver, spleen and heart.
16. A method of detecting presence of a cryptic intracellular
infection by an intracellular pathogen, comprising contacting cells
suspected of being cryptically infected with a test agent which
stimulates the cryptic form of the intracellular pathogen to enter
an active phase, determining the presence or absence of the
intracellular pathogen.
17. The method of claim 16 wherein the presence of the pathogen is
determined by detecting the active phase of the pathogen by visual
detection or by reverse transcriptase PCR.
18. The method of claim 17 wherein the active phase is determined
by visual detection of inclusion bodies or immunochemical detection
of chlamydial antigen.
19. The method of claim 16 wherein the pathogen is detected by
determining the presence or absence of a gene for that
intracellular pathogen.
20. The method of claim 16 wherein the test agent is a single agent
or plurality of different test agents.
21. The method of claim 16 wherein the intracellular pathogen is
Chlamydia.
22. The method of claim 21 wherein the Chlamydia is Chlamydia
pneumoniae.
23. The method of claim 22 wherein the gene for Chlamydia
pneumoniae is MOMP.
24. The method of claim 16 wherein the presence or absence of the
gene for the intracellular pathogen is determined by a nucleic acid
amplification technique.
25. The method of claim 24 wherein the nucleic acid amplification
technique is PCR.
26. An assay for identifying an agent which is capable of
inhibiting or eliminating intracellular pathogen infection,
comprising the steps of: a) preparing tissue culture cells infected
with an intracellular pathogen in the absence of cycloheximide; b)
allowing the intracellular pathogen to replicate; c) adding a test
agent; d) isolating intracellular pathogen nucleic acid from the
cells; e) amplifying the intracellular pathogen nucleic acid by a
nucleic acid amplification technique; and f) evaluating the
presence or absence of amplified intracellular pathogen nucleic
acid; wherein the absence of amplified intracellular pathogen
nucleic acid is indicative that the agent is capable of inhibiting
or eliminating intracellular pathogen infection.
27. The assay of claim 26 wherein the intracellular pathogen is
Chlamydia.
28. The assay of claim 27 wherein the Chlamydia is Chlamydia
pneumoniae.
29. The assay of claim 26 wherein the amplification technique is
PCR.
30. A method of identifying cells containing cryptic form of an
intracellular pathogen, comprising the steps of: a) treating
cultured cells, thought to be infected with an intracellular
pathogen, with a disulfide reducing agent; b) subjecting cultured
cells to protease digestion; c) exposing cells to appropriate
polymerase, dNTPs and primers for DNA amplification of a gene
encoding a protein of the intracellular pathogen; d) exposing the
cells to a reporter molecule enzyme; e) exposing the cells to an
appropriate substrate for the reporter enzyme; and f) determining
the presence of cryptic form of Chlamydia by visualizing the
amplified DNA encoding the protein.
31. The method of claim 30 wherein the intracellular pathogen is
Chlamydia.
32. The method of claim 31 wherein the Chlamydia is Chlamydia
pneumoniae.
33. The method of claim 32 wherein the primers of step c) are
CHLMOMPDB2 and CHLMOMPCB2.
34. A method of identifying cells containing cryptic form of an
intracellular pathogen comprising the steps of: a) treating
cultured cells, thought to be infected with an intracellular
pathogen, with guanidine thiocyanate; b) exposing cells to
appropriate polymerase, dNTPs and primers for DNA amplification of
a gene encoding a protein of the intracellular pathogen; c)
exposing the cells to a reporter molecule enzyme; d) exposing the
cells to an appropriate substrate for the reporter enzyme; and e)
determining the presence of cryptic form of the pathogen by
visualizing the amplified DNA encoding the protein.
35. The method of claim 34 wherein the intracellular pathogen is
Chlamydia.
36. The method of claim 35 wherein the Chlamydia is Chlamydia
pneumoniae.
37. The method of claim 36 wherein the primers of step c) are
CHLMOMPDB2 and CHLMOMPCB2.
38. An assay for identifying an agent which is effective against
cryptic form of an intracellular pathogen, comprising the steps of:
a) treating cultured cells grown in the absence of cycloheximide,
thought to be infected with an intracellular pathogen, with a
disulfide reducing agent; b) allowing the pathogen to replicate; c)
adding a test agent; d) subjecting cultured cells to protease
digestion; e) exposing cells to appropriate polymerase, dNTPs and
primers for DNA amplification of a gene encoding a protein of the
intracellular pathogen; f) exposing the cells to a reporter
molecule enzyme; g) exposing the cells to an appropriate substrate
for the reporter enzyme; h) determining the presence of cryptic
form of the pathogen by visualizing the amplified DNA encoding the
protein; wherein the absence of amplified nucleic acid is
indicative that the agent is effective against cryptic form of the
pathogen.
39. The assay of claim 38 wherein the intracellular pathogen is
Chlamydia.
40. The assay of claim 39 wherein the Chlamydia is Chlamydia
pneumoniae.
41. The assay of claim 40 wherein the appropriate primers of step
a) are CHLMOMPDB2 and CHLMOMPCB2.
42. A method for identifying an agent or combination of agents
which is capable of inhibiting intracellular pathogen infection in
a patient or an animal, comprising the steps of: a) culturing
intracellular pathogen infected cells obtained from the infected
patient or animal; b) allowing the intracellular pathogen to
replicate; c) adding one or more test agents; d) exposing the cells
to protease and reducing agents, or guanidine thiocynate to isolate
intracellular pathogen nucleic acid; e) amplifying the nucleic acid
by a nucleic acid amplification technique; and f) evaluating the
presence or absence of amplified nucleic acid; wherein the absence
of amplified nucleic acid is indicative that the agent or
combination of agents is capable of inhibiting pathogen
infection.
43. The method of claim 42 wherein the intracellular pathogen is
Chlamydia.
44. The method of claim 43 wherein the Chlamydia is Chlamydia
pneumoniae.
45. The method of claim 44 wherein the primers of step d) are
CHLMOMPDB2 and CHLMOMPCB2.
46. The method according to claim 42 wherein at least two or more
probes are used to reduce false positives.
47. A method for determining the presence of cryptic form of an
intracellular pathogen, comprising exposing cells, thought to be
infected with cryptic form of the pathogen, with a reducing agent,
for a period of time sufficient to rupture the cryptic cells and
thereby release nucleic acids contained therein; and determining
the presence or absence of the intracellular parasite by
determining the presence or absence of a gene for that
intracellular parasite.
48. The method of claim 47 herein the intracellular pathogen is
Chlamydia.
49. The method of claim 48 wherein the Chlamydia is Chlamydia
pneumoniae.
50. The method of claim 49 wherein the gene for Chlamydia
pneumoniae is MOMP.
51. The method of claim 47 wherein the presence or absence of the
gene for the intracellular pathogen is determined by a nucleic acid
amplification technique.
52. The method of claim 51 wherein the nucleic acid amplification
technique is PCR.
53. An agent or combination of agents identified by any one of the
methods described herein.
54. A method for eliminating a Chlamydia infection, comprising
contacting infected cells with a combination of isoniazid,
metronidazole and penicillamine in an amount and for a period of
time effective to eliminate the Chlamydia therefrom.
55. The method of claim 54 wherein the period of time effective to
eliminate the Chlamydia infection is measured by determining the
presence or absence of a Chlamydial gene in the cells; wherein the
absence of the gene is indicative that the infection has been
eliminated.
Description
RELATED APPLICATION
[0001] This application claims priority to U.S. patent application
Ser. No. 08/911,593, filed on Aug. 14, 1997, the entire teachings
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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).
[0004] 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).
[0005] 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.
[0006] 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.
[0007] 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
[0008] 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.
[0009] 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.
[0010] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0011] This invention pertains to novel approaches for the
susceptibility testing of Chlamydia species that are necessitated
by the complex life cycle of the chlamydial pathogen as well as by
its diverse, extensive, and heretofore unappreciated ability to
cause chronic, cryptic, and persistent systemic infections that are
refractory to short duration therapy with conventional single
agents. The inventors have discovered that successful eradication
of chronic/systemic chlamydial infections can be predicted by using
the described unique methods for in vitro and in vivo
susceptibility testing.
[0012] The invention is based upon the discovery that current
susceptibility testing methods for Chlamydiae do not accurately
predict the ability of antimicrobial agents to successfully and
totally eradicate chronic chlamydial infections. This is because
the current susceptibility testing methods measure only replication
of chlamydia and ignores the well-known "cryptic phase" (28-33) in
which Chlamydiae are not actively replicating. Moreover, it has
also been discovered that the so-called "cryptic phase" of
Chlamydiae includes multiple and different phases. The following
are phases of the chlamydial life cycle in which the Chlamydiae are
not replicating: an initial intranuclear phase in which elementary
bodies (EBs) transition to reticulate bodies (RBs), an
intracytoplasmic phase in which there is a transition of the RB
phenotype to the EB phenotype, an intracytoplasmic phase with a
nonreplicating, but metabolizing RB, and
intracellular/extracellular EB phases in which there is neither
replication nor metabolsim. In order to assess the cumulative and
long term effect of antimicrobial therapy on these multiple life
phases, unique in vitro and in vivo susceptibility test methods
have been developed and are described herein.
[0013] The term "susceptibility" as used herein is intended to mean
a physiological response of an organism to an environmental or
chemical stimuli. The desired physiological response to stimuli is
one which adversely affects the pathogen's viability to replicate
or reside within the host cell and, ideally, would result in the
complete elimination (i.e., death) of that pathogen.
[0014] In Vitro Methodology
[0015] One aspect of the invention pertains to methods for
evaluating the susceptibility of the distinct phases and stages of
the life cycle of Chlamydia, to a particular agent(s), particularly
the cryptic phase, since prior techniques have failed, heretofore,
to appreciate the need for drugs that can clear infected cells of
cryptic Chlamydia. A preferred drug screening method which
accomplished this objective utilizes tissue culture cells, in the
absence of cycloheximide in order to encourage cryptic infection.
Cryptic infection is less likely to occur in cells used in standard
cell culture susceptibility techniques because Chlamydia in
cycloheximide-paralyzed cells need not compete with the host cell
for metabolites and hence are encouraged to replicate.
[0016] In a preferred embodiment, the cells are grown in a culture
medium in which chlamydial forms have been inactivated as described
in U.S. Ser. No. ______, entitled "Chlamydia-Free Cell Lines and
Animals", filed concurrently herewith; the entire teachings of
which are incorporated herein by reference.
[0017] The in vitro method uses standard tissue culture cells, but
without the addition of cycloheximide. Moreover, the chlamydiae are
allowed to replicate for several days prior to the addition of at
least one test agents. A "test agent" can be any compound to be
evaluated as an antichlamydial agent for its ability to
significantly reduce the presence of Chlamydia in living cells. For
example, a test agent can include, but is not limited to,
antibiotics, antimicrobial agents, antiparasitic agents,
antimalarial agent, disulfide reducing agents and antimycobacterial
agents. The test agent(s) is/are replaced when needed for the
duration of the incubation time (days to weeks) to ensure that the
test agent is present and has not been otherwise degraded.
Antimicrobial agent(s) (test agent) is then added to the
replicating cells. The antimicrobial agents/growth medium are
periodically replaced for the duration of the incubation time,
which is preferably weeks rather than days. Finally, the end point
after the prolonged incubation time is the complete absence of
chlamydial DNA, as determined by a nucleic acid amplification
technique, such as the polymerase chain reaction (PCR) methodology.
Standard nucleic acid amplification techniques (such as PCR) are
used to ascertain the presence or absence of signal for chlamydial
DNA encoding MOMP or other unique Chlamydia protein to determine
whether the test agent or combination of agents is/are effective in
reducing Chlamydia infection. The loss of signal (i.e., below the
detectable level of the nucleic acid amplification technique) in
cells with antibiotic(s) versus its presence in controls is an
indication of efficacy of the agent or combination of agents
against Chlamydia.
[0018] Accordingly, the susceptibility test of this invention can
be used to identify an agent or agents which are effective against
any particular species of Chlamydia and can be used to identify
agent(s) effective against the cryptic form of the pathogen, i.e.,
is capable of inhibiting or eliminating the cryptic form of the
pathogen. Agents that are effective against Chlamydia, as
ascertained by the susceptibility testing protocols described
herein, can be used as part of a therapy for the management of
Chlamydia infections. Suitable therapeutic protocols are described
in detail below, with a particular focus on targeting agents toward
specific stages of the chlamydial life cycle.
[0019] The methods described herein are unique because they
evaluate the activity of antimicrobial agents in the absence of
cycloheximide which provides a more clinically relevant
intracellular milieu. For example, any energy-dependent host cell
membrane pumps which might move antimicrobial agents in or out of
the cell are inactivated by the use of cycloheximide. The methods
described herein are unique because they utilize culture medium
which has previously been inactivated. The methods are also unique
because they measure the effect of a prolonged duration of exposure
to the antimicrobial agent(s) after the intracellular infection by
chlamydiae has become established. Finally, the method is unique
because it measures the presence/absence of chlamydial DNA as the
endpoint, for example by measuring PCR signal. By using complete
eradication of chlamydial DNA as an endpoint, the susceptibility
test confirms that all phases of Chlamydiae have been eradicated as
opposed to merely a temporary halt in replication.
[0020] When PCR is the preferred methodology used to evaluate assay
endpoint, the PCR method can be enhanced by the unique application
of a reducing agent, such as dithiothreitol (DTT), in order to
uncoat chlamydial EBs and hence allow exposure of the DNA. In other
words, DTT permits the EB coating to rupture. By using an assay for
DNA in which EBs are specifically uncoated, the susceptibility test
endpoint assesses the presence or absence of EBs as well as the
presence or absence of both replicating and nonreplicating RBs.
Thus, this approach for chlamydial susceptibility testing allows
quantitative antimicrobial susceptibility assays of single and
combination agents in which the cumulative effect of the agent(s)
on the complete eradication of all life phases is measured.
Examples of results obtained with this in vitro method are
described below.
[0021] In one embodiment, a suitable nucleic acid assay for
identifying agents effective against the cryptic form of chlamydia
comprises, in the presence of agent(s) to be tested, subjecting
cultured cells to protease/reducing agent (e.g., dithiotreitol) and
protease digestion or guanidine isothiocyanate (also known as
guanidine thiocyanate) for a prescribed period of time; extracting
DNA from the treated solution; exposing DNA to appropriate
polymerase, dNTPs and primers for DNA amplification of MOMP or
other protein of the Chlamydia species; and determining the
presence or absence of amplified DNA by visualizing the ethidium
bromide treated DNA product by gel electrophoresis, for example. In
particular embodiments, the Chlamydia species is C. pneumoniae and
the appropriate primers are CHLMOMPDB2 and CHLMOMPCB2.
[0022] The invention further relates to a method of identifying
cells containing the cryptic form of a Chlamydia species by a
nucleic acid amplification technique (e.g., PCR) comprising
subjecting cultured cells to protease digestion; stopping protease
activity; exposing cells to appropriate heat-stable DNA polymerase,
dNTPs and labeled primers (e.g., 3'-biotin labeled, 5'-biotin
labeled) for amplification of DNA encoding MOMP of the Chlamydia
species; washing the cells; exposing the cells to a reporter
molecule (e.g., strepavidin-conjugated signal enzyme); exposing the
cells to an appropriate substrate for the reporter molecule (e.g.,
conjugated enzyme); and visualizing the amplified DNA encoding MOMP
by visualizing the product of the reaction.
[0023] The invention pertains to a method of identifying cells
containing the cryptic form of Chlamydia. The method comprises
treating cultured cells, thought to be infected with Chlamydia,
with a disulfide reducing agent; subjecting cultured cells to
protease digestion; exposing cells to appropriate polymerase, dNTPs
and primers for DNA amplification of nucleic acid encoding of a
chlamydial protein; exposing the cells to a reporter molecule
enzyme; exposing the cells to an appropriate substrate for the
reporter enzyme; and determining the presence of the cryptic form
of Chlamydia by visualizing the amplified DNA encoding a chlamydial
protein. Preferably the amplification technique is PCR and the
primers are CHLMOMPDB2 and CHLMOMPCB2 of Chlamydia pneumoniae.
[0024] A similar method can be used as an assay for identifying an
agent which is effective against the cryptic form of Chlamydia.
Accordingly, the method comprises treating cultured cells grown in
the absence of cycloheximide, thought to be infected with
Chlamydia, with a disulfide reducing agent; allowing the Chlamydia
to replicate; adding a test agent; subjecting cultured cells to
protease digestion; exposing cells to appropriate polymerase, dNTPs
and primers for DNA amplification of a gene encoding chlamydial
protein; exposing the cells to a reporter molecule enzyme; exposing
the cells to an appropriate substrate for the reporter enzyme; and
determining the presence of cryptic form of Chlamydia by
visualizing the amplified DNA encoding a chlamydial protein, such
as MOMP.
[0025] A detailed description of primers, PCR techniques and other
methodologies useful for the present invention are provided in U.S.
patent application Ser. No. 08/911,593, filed Aug. 14, 1997,
entitled "Diagnosis and Management of Infection Caused by
Chlamydia" by William M. Mitchell and Charles W. Stratton; U.S.
patent application Ser. No. ______ entitled "Chlamydia Free Cell
Lines Animals" (Attorney's Docket No. VDB98-03), filed concurrently
herewith; and U.S. patent application Ser. No. ______ entitled
"Identification of Antigenic Peptide Sequences" (Attorney Docket
No. VDB98-01), filed concurrently herewith; the entire teaching of
these applications are incorporated herein by reference.
[0026] In Vitro Susceptibility Testing Results
[0027] Two week exposure of single agents including the
fluoroquinolone, ofloxacin, and the macrolide, clarithromycin, at 1
.mu.g/ml failed to clear HeLa cells in culture of a detectable PCR
signal for the MOMP gene of Chlamydia pneumoniae. In contrast,
triple agents consisting of isoniazid (INH), metronidazole, and
penicillamine (1 .mu.g/ml each) resulted in no detectable PCR
signal (Table 1). None of these agents, effective in the triple
combination, is currently recognized as an anti-chlamydial
agent.
[0028] Table 2 provides the results of an expanded study of
antimicrobial susceptibilities at two different concentrations of
antimicrobial agents, used alone and in combination, when exposed
to the antimicrobial agents for two weeks. In addition to the
agents already mentioned, minocycline, doxycycline, rifampin and
sulfamethoxizole/trimethoprim, at all concentrations tested, failed
to clear the PCR signal for chlamydial MOMP. Only the triple
combination of isoniazid, metronidazole and penicillamine cleared
the PCR signal. The triple combination was effective at both low
and high concentrations. Table 2 also demonstrates the effect of a
4 week exposure with the same expanded series of antimicrobial
agents alone and in combination. A number of triple combinations of
antimicrobial agents resulted in cell cultures in which the PCR
signal for the chlamydial MOMP gene could not be detected.
1TABLE 1 Susceptibility to Antibiotics for Cryptic Chlamydia
pneumoniae Cultured in HeLa Cells.sup.a Antibiotic Conc (.mu.g/ml)
PCR.sup.b Ofloxacin 1 positive Clarithromycin 1 positive INH 1
positive Metronidazole 1 positive Penicillamine 1/1 positive INH +
1/1/4 negative Metronidazole + 0 positive Penicillamine Control
.sup.aCultured in the presence of the indicated antibiotic(s), but
with no cycloheximide. Media changes at 48-72 hours. .sup.bAnalysis
following 2 week exposure to antimicrobial agents.
[0029]
2TABLE 2 Susceptibility to Antibiotics for Cryptic Chlamydia
pneumoniae Cultured in HeLa Cells.sup.a by PCR Conc PCR PCR
Antibiotic (.mu.g/ml) 2 week 4 week Minocycline 1 pos pos
Doxycycline 1 pos pos Isoniazide 1 pos pos TMP/SMZ.sup.b 100 pos
pos Minocycline + Metronidazole + penicillamine 1/1/4 pos pos
Doxycyclin + Metronidazole + penicillamine 1/1/4 pos neg Isoniazid
+ Metronidazole + penicillamine 1/1/4 neg neg TMP/SMZ +
Metronidazole + penicillamine 100/1/4 pos neg Metronidazole 0.25
pos pos Clarithromycin 0.25 pos pos Rifampin 0.25 pos pos Ofloxacin
0.25 pos pos Minocycline 0.25 pos pos Doxycycline 0.25 pos pos
TMP/SMz + Metronidazole 25/0.25 pos Ofloxacin + Metronidazole
0.25/0.25 pos pos Rifampin + Metronidazole + penicillamine
0.25/0.25/4 pos pos Rifampin + Metronidazole + Ofloxacin
0.25/0.25/0.25 pos pos Clarithromycin + Metronidazole +
penicillamine 0.25/0.25/1 pos neg Doxycycline + Metronidazole +
penicillamine 0.25/0.25/1 pos pos Minocycline + Metronidazole +
penicillamine 0.25/0.25/1 pos neg Isoniazid + Metronidazole +
penicillamine 0.25/0.25/1 neg neg TMP/SMZ 0.25 pos Rifampin +
Metronidazole 0.25/0.25 pos None 0 pos pos .sup.aCultured in the
presence of the indicated antibiotics, but with no cycloheximide.
Media changes at 48-72 hours. pos = positive; neg = negative
.sup.bTMP/SMZ = trimethoprim/sulfamethoxazole
[0030] In Vivo Methodology
[0031] In another aspect of the invention, the susceptibility test
can be used to evaluate the status of a human or animal undergoing
therapy for the management of Chlamydia infection. For example, a
biological material is isolated from the human or animal undergoing
combination therapy. The biological material is treated such that
the Chlamydia is isolated therefrom. This chlamydial isolate is
allowed to infect Chlamydia free cells. These infected cells are
then exposed to the combination of agents being used in the
individual undergoing combination therapy. Alternatively, the
individual's serum containing the antimicrobial agents can be added
to the infected cells as a "serum bactericidal test" for
intracellular chlamydial infection. Methods for producing Chlamydia
free cells are described in U.S. patent application Ser. No.
______, entitled "Chlamydia Free Cell Lines and Animals"
(Attorney's Docket No. VDB98-03), filed concurrently herewith; the
entire teachings of which are incorporated herein by reference.
[0032] The in vivo method uses the murine model although other
animals such as rats or rabbits can be used. In this method, mice
(or any other animal) are inoculated intranasally with
2.times.10.sup.5 chlamydial EBs per ml. The inventors have
confirmed the work of Yang and colleagues (15) in which intranasal
inoculation of chlamydial EBs results in systemic dissemination
and, in particular, causes infection of the spleen. The inventors
have discovered that this systemic dissemination also results in
the presence of EBs in the blood of the mice. Therefore,
infectivity can be measured by blood culture or by serum/whole
blood PCR for chlamydial DNA. Systemic infection is also confirmed
and monitored by the presence of elevated IgM and IgG antibody
titers. After the systemic murine infection has been established,
antimicrobial agents are given to the mice. This is most easily
done by adding the antibiotics to the drinking water. The effect of
antichlamydial therapy is monitored by serum/whole blood PCR. When
the serum/PCR assay suggests eradication of chlamydiae from the
bloodstream, the mice are sacrificed and PCR for chlamydial DNA is
done on lung, heart, liver, and spleen homogenates. This method is
unique because it measures the complete eradication of all life
forms of chlamydiae in known murine target organs for chlamydial
infection. This in vivo susceptibility method has revealed, for
example, that antimicrobial therapy with the triple agents, INH,
metronidazole and penicillamine, can completely eradicate C.
pneumoniae from infected mice in four months. Moreover, following
complete eradication of chlamydiae, multiple attempts to reinfect
these cured mice via intranasal inoculation have proven
unsuccessful. This suggests that effective therapy and complete
eradiaction results in the development of protective immunity, and
that effective therapy is therefore a way to create effective
immunity.
[0033] Performing PCR for chlamydial DNA on homogenates of other
organ systems can be used to determine the effectiveness of
particular antibiotic combinations in eradicating chlamydial
infection in those organ systems. Establishment of prior chlamydial
infection of those systems can be done by either biopsy or
antibody-enhanced radiological imaging. Alternatively, prior
infection can be determined statistically by performing PCR for
chlamydial DNA on homogenates of the same organ systems in a
similarly inoculated but untreated control population.
Organ-specific susceptibility is determined by comparing rates of
positive PCR assays in the control and treated populations.
[0034] An alternative or complementary method of determining the
presence of cryptic chlamydial infections in an animal or cell
culture is to expose the culture to chlamydia-stimulating
compounds. Such compounds include (but are not limited to)
cycloheximide, corticosteriods (such as prednisone) and other
compounds which are known to stimulate reactivation of cryptic
intracellular infections, and disulfide reducing agents (such as
dithiotreitol) and other chemicals which cause EBs to turn into
RBs. Once the cryptic forms have entered a more active phase, they
can be detected using standard detection techniques such as visual
detection of inclusion bodies, immunochemical detection of
chlamydial antigen, or reverse transcriptase-PCR.
[0035] Although the foregoing description is directed toward
Chlamydia, it is merely for exemplary purposes and is not intended
to limit the invention thereto. The invention therefore is relevant
for other to obligate intracellular pathogens. For example,
pathogens that must be in an intracellular location in order to
replicate, include but are not limited to prions, viruses,
Chlamydiae spp., Mycoplasma spp., Ehrilichia spp., Rickettsia spp.,
Bartinella spp., Borrelia spp., Toxoplasma gondii, Leishmania spp.
and Trypanosomes (e.g., Malaria). Additionally, included are
pathogens that are able to survive in an intracellular location and
can find a physiologic advantage to do so, for example, Legionella
spp., Salmonella spp., Listeria spp., Histoplasma spp., Yersinia
spp. and Mycobacteria spp. Intracellular pathogens that are able to
utilize selective intracellular locations to enhance survivability
and/or pathogenics, are embraced in this invention and include but
are not limited to Neisseria spp., Staphylococcus spp., Hemophilus
spp., Escherichia coli, Candida spp. and Torulopsis spp.
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[0069] Equivalents
[0070] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *