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Journal of Clinical Microbiology, July 2000, p. 2591-2594, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Failure To Detect Chlamydia pneumoniae
in the Late-Onset Alzheimer's Brain
Robert H.
Ring1,* and
Joseph M.
Lyons2
Division of Neurosciences, Beckman Research
Institute at the City of Hope,1 and
Department of Infectious Diseases, City of Hope National
Medical Center,2 Duarte, California 91010
Received 31 January 2000/Returned for modification 22
March 2000/Accepted 25 April 2000
 |
ABSTRACT |
Epidemiological studies have yet to identify a single cause for the
most common late-onset form of Alzheimer's disease. The common
respiratory pathogen Chlamydia pneumoniae recently has been
implicated as a risk factor for this form of Alzheimer's disease. Were
this true, there would be a dramatic shift in current paradigms of Alzheimer's disease research and treatment. In the absence of published confirmation, we obtained postmortem brain tissue
from late-onset Alzheimer's disease patients (n = 15)
and representative controls (n = 5) and extracted DNA
from up to six separate brain regions in each instance, including those
areas particularly relevant to Alzheimer's disease neuropathology.
Each sample of DNA (n = 101) was assayed five times or
more for the presence of C. pneumoniae DNA using a
nested-PCR protocol targeting a species-specific gene sequence coding
for the major outer membrane protein of this organism. We were unable
unequivocally to detect C. pneumoniae in any of the 101 samples tested by PCR and failed to culture the organism from tissue
samples. We conclude that C. pneumoniae is neither strongly
nor uniquely associated with the neuropathology seen in late-onset
Alzheimer's disease.
| |
INTRODUCTION |
Alzheimer's disease (AD) is a
neurodegenerative disorder characterized behaviorally by the
progressive loss of memory and disorder of other cognitive functions.
Most cases are of the nonfamilial, late-onset type for which a specific
cause(s) has yet to be established, although a number of risk factors
have been identified (8). A recent report has raised the
intriguing possibility that Chlamydia pneumoniae, a common
human respiratory pathogen, may be a risk factor in late-onset AD
(2). C. pneumoniae was reported to be detectable
by PCR in 17 of the 19 (90%) late-onset AD brains examined, compared
to 1 of 19 (5%) controls. These results were unequivocal in two assays
and were confirmed in selected subsets of these samples by electron
microscopy, immunoelectron microscopy, immunohistochemistry, reverse
transcription-PCR, and culture analysis. Were this to be the case,
there would be a dramatic shift from current paradigms employed in AD
research and treatment.
Since the presence of C. pneumoniae in AD brains has not
been confirmed, an independent investigation was performed to examine the extent of C. pneumoniae presence in the brains of
late-onset AD patients. Postmortem brain tissue from late-onset AD and
appropriate controls was screened for evidence of C. pneumoniae DNA using PCR targeting a species-specific gene
sequence of the major outer membrane protein (MOMP), as
previously published (4), and modified since publication by
the original authors (personal communication). Attempts were also made
to culture the organism from tissue homogenates.
| |
MATERIALS AND METHODS |
Clinical tissue specimens.
Postmortem tissue samples from
the brains of late-onset AD patients and appropriate controls were
obtained from the Alzheimer's Disease Research Center Neuropathology
Core, USC School of Medicine. All cases fulfilled the neuropathologic
criteria for definite AD as defined by the Consortium to Establish a
Registry for Alzheimer's Disease. Samples were frozen following
autopsy (the average postmortem intervals for controls and AD samples
were 6.3 and 5.9 h, respectively) and stored at 
70°C until DNA
extraction. Average ages of control and AD patients at time of death
were 86.2 and 77.0 years, respectively. Stringent precautions were
employed during the processing of clinical specimens to avoid the
possibility of contamination while performing dissections, nucleic acid
extractions, and culturing from clinical samples in separate
laboratories at different times.
Extraction of DNA from tissue.
DNA was extracted from tissue
using hot buffered phenol. Briefly, 50 mg of frozen central nervous
system (CNS) tissue was placed in a 7-ml Dounce glass homogenizer
containing 0.5 ml of lysis buffer (100 mM NaCl, 10 mM Tris-HCl, 25 mM
EDTA, and 0.5% sodium dodecyl sulfate; final pH of 8.0) and
homogenized briefly before adding 1.0 ml of phenol (65°C, pH 8.0).
The sample was then homogenized for another 2 min, transferred to a
sterile 1.7-ml tube, and centrifuged at 12,000 rpm for 20 min. Aqueous
phase was carefully removed and washed twice with phenol at room
temperature, which was followed by two washes with chloroform. DNA was
precipitated with three volumes of ethanol (20°C) and centrifuged
at 12,000 rpm for 20 min. Pellets were washed twice with 70% ethanol,
air dried, and resuspended in Tris-EDTA (pH 8). This method differs from the reference cited in the original paper describing the method
(4). This modification was conveyed to us to be the currently preferred procedure by the senior author of the original paper (personal communication). Our own comparison of the two methods
confirmed there to be improvement in DNA yield with the above
modification. Spectrophotometric analysis was used for quantitative determination and standardization of DNA concentrations.
PCR.
A nested PCR was performed five or more times using DNA
samples extracted from each patient, with 1 µg of DNA per reaction mixture. The primers targeted a species-specific sequence of the MOMP
gene of C. pneumoniae (13) and were synthesized
by the City of Hope DNA Synthesis Facility according to sequences
provided by Alan Hudson. The primers used were outer forward
(5'-TATTATCCGCCGCATTTG), corresponding to bases 26 to 43 of the
MOMP gene; outer reverse (5'-GAGGTGTCTGTGTAAAGTTC), corresponding to
bases 560 to 541; nested forward (5'-TACAATATGGGAAGGTGCTGC),
corresponding to bases 114 to 134; and nested reverse
(5'-TGAACGCTGTAGAGTTTCC), corresponding to bases 457 to 439. These sequences differ from those published in the original paper, but
represent those currently used in Dr. Hudson's laboratory (personal communication).
The PCR cocktail used contained 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 0.001% (wt/vol) gelatin; and 0.25 U of AmpliTaq Gold DNA polymerase (catalog no. N808-0241; Perkin-Elmer).
Thermocycling was performed on an MJ Research PT-100 device with a hot
bonnet. The PCR program consisted of 95°C for a 10-min hot start; 35 cycles of denaturation at 95°C, annealing at 55°C, and extension at
72°C (each for 1 min); and a final extension at 72°C for 5 min. A
2.5-µl aliquot from the first reaction mixture was added to a second reaction cocktail and was identical except for the substitution of
nested primers. Products were run on 2% agarose gel and
Tris-borate-EDTA running buffer and were visualized by ethidium bromide
staining. The PCR assay of each sample was repeated a minimum of five
times. Products were isolated, and sequences were verified on an ABI prism sequencer. Reaction setup, thermocycling, and product analysis were performed in separate laboratories to avoid problems associated with amplimer contamination.
Propagation of Chlamydia strains.
Several strains of
Chlamydia were propagated and enumerated for use in control
experiments. C. pneumoniae AR39 (ATCC 53592), C. pneumoniae CM-1 (ATCC VR-1360), Chlamydia trachomatis
serotype D (ATCC VR-885) and Chlamydia psittaci (ATCC
VR-351) were propagated and titrated in cyclohexamide (1 µg/ml)-inhibited HEp-2 cells (ATCC CCL-23) grown in 20 mM HEPES
buffered and glutamine containing (1 mM) Eagle's minimal essential
medium supplemented with 10% heat-inactivated fetal bovine serum and
0.45% glucose. C. pneumoniae inclusions were detected and
enumerated using standard indirect enzyme-linked immunosorbent assay
techniques that employed a species-specific anti-C.
pneumoniae monoclonal antibody (DAKO Corp. M660001).
Culture of C. pneumoniae from brain tissue.
Homogenates from PCR-positive brain samples and identical regions from
control brains were cultured for C. pneumoniae in the human
monocyte cell line THP-1 (ATCC TIB-202) by the method reported in the
original paper (4) with slight modification. Approximately 0.5 g of brain tissue, in 2.0 ml of THP-1 growth medium (RPMI 1640 medium [Irvine Scientific, Santa Ana, Calif.] supplemented with 10%
heat-inactivated fetal bovine serum [Omega Scientific, Tarzana,
Calif.], 10 mM HEPES, 1 mM sodium pyruvate, 1 mM glutamine [all from
Irvine Scientific], and 0.45% glucose [Mallinckrodt Baker, Paris,
Ky.]), were subjected to three freeze-thaw cycles with tissues
homogenized and sonicated between cycles. Four hundred microliters of
homogenate was mixed with 3 × 106 THP-1 cells
suspended in 2 ml of fresh growth medium, centrifuged for 30 min at
500 × g, diluted to a 10-ml volume with medium, transferred to 25-cm2 tissue culture flasks, and incubated
for 72 h at 37°C in 5% CO2. Following initial
culture, 1 ml of culture was used for secondary passage in 3 × 106 THP-1 cells as described above, and 4 ml was frozen at
70°C. The remaining 5 ml of primary culture material was
centrifuged at 500 × g for 30 min, separated into
pellet and supernatant, and frozen at 70°C until analyzed by PCR.
Secondary culture material was processed as described above, with one
half frozen neat and the other half processed for PCR.
Serving as positive controls, companion aliquots of brain tissue
homogenates were seeded with approximately 100 infection-forming
units
(IFU) of
C. pneumoniae (AR39) prior to initial
centrifugation
with THP-1 cells and were processed alongside the test
aliquots.
| |
RESULTS |
Specificity and sensitivity of PCR.
The PCR assay employed
distinguished two separate strains of C. pneumoniae from
C. trachomatis and C. psittaci as shown in Fig.
1. Both the outer and nested PCR products
are shown. The product of the first (outer) amplification is 538 bp,
and the second (nested) amplification produced a product of 344 bp.
Each product was verified by sequence analysis (data not shown).
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|
FIG. 1.
Species specificity of PCR assay. Both the outer and
nested primer pairs specifically amplify two strains of C. pneumoniae (C.pn) DNA but do not amplify DNA from C. trachomatis (C.t) or C. psittaci (C.ps). A 100-bp
ladder was run (lanes labeled 100) alongside PCR products for size
determination.
|
|
To estimate the sensitivity of our PCR assay, a dilution series of
C. pneumoniae DNA (10
4 to 10
4 IFU)
was added to 1 µg of human genomic DNA prior to performing
nested PCR
(Fig.
2). In five separate PCRs we were
consistently
able to detect between 0.01 and 0.001 IFU within this
1-µg background
of nontarget nucleic acids. This degree of
sensitivity is in agreement
with previously reported assays (
5,
10,
11). The detection
of less than 1 IFU demonstrates the
ability of the assay to detect
both cultivatable and replicating forms
of
C. pneumoniae.
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FIG. 2.
Range of detection. Known amounts of C. pneumoniae DNA (103 to 106 IFU) were
spiked into a constant background of human genomic DNA (1 µg) and
assayed by PCR. (A) The first round of amplifications with the outer
primer pair demonstrates the ability of the assay to detect as few as
0.1 IFU. (B) The second round of amplifications using the nested primer
pair extends this range to 0.001 to 0.0001 IFU. A 100-bp ladder was run
(lanes labeled 100) alongside PCR products for size determination.
|
|
To obviate the potential sampling errors associated with a
low-copy-number amplification of
C. pneumoniae from within
our
DNA samples, PCRs were repeated separately a minimum of five times.
The DNA used in each PCR (~1 µg) was derived from roughly 150,000
cells (
9), bringing the maximum sensitivity of the combined
five PCRs down to 0.01 to 0.001 IFU per 750,000
cells.
Analysis of extraction.
Extraction of DNA from clinical
samples for use in PCR assays can be problematic because of the
historically recognized problems generally associated with recovery
efficiencies of different DNA extraction methods as well as the
copurification of PCR inhibitors (e.g., lipids, Ca2+,
acidic polysaccharides, etc.) (10, 15).
To determine the efficiency of extraction we added a dilution series of
C. pneumoniae (10
4 to 10
4 IFU) to
control brain tissue prior to homogenization and extraction
of DNA and
then assayed 1 µg of the recovered nucleic acids by
PCR for
C. pneumoniae (data not shown). We were able to detect
a minimum of 1 IFU of
C. pneumoniae following extraction. This
sensitivity
of the assay is actually much greater, because the
1 µg of input DNA
represented the entire DNA sample recovered.
The average yield of total
DNA per extraction was roughly 30 µg,
bringing the range of detection
following extraction down to approximately
0.03 IFU. This is similar to
the detection sensitivity of the
PCR assay reported above and indicates
that there is minimal loss
of DNA or that significant copurification of
inhibitors had not
occurred during our
extraction.
PCR of patient samples.
Amplification of potentially
low-copy-number targets within a disproportionately larger background
of host DNA requires modifications of traditional PCR in order to
ensure specificity and efficiency of the reaction (9). A
nested PCR following the first round of amplification was employed to
increase the efficiency of identifying C. pneumoniae. Nested
PCR involves a second round of amplification with primers internal to
the original primer pair. This technique gives a built-in confirmation
of the outer products and is an advantage for low-copy-number
amplifications because of its ability to dilute the original material
along with any inhibitory substances (1). The use of
AmpliTaq Gold DNA polymerase allowed the integration of a hot start
into the amplification (3). A hot start can significantly
improve the specificity, sensitivity, and yield in PCR by eliminating
the formation of misprimed products and primer-oligomers often formed
during pre-PCR at temperatures lower than the optimal annealing
temperature (6).
DNA samples from six separate brain regions of each patient were
assayed for
C. pneumoniae by PCR five or more times. These
regions included areas relevant to AD (e.g., the hippocampus,
frontal
cortex, and parietal cortex). For a case to be considered
positive, the
PCR results had to be unequivocally positive for
at least one of the
regions sampled. Our PCR results were uniformly
negative for
C. pneumoniae in both control and AD samples (Table
1; Fig.
3).
Although three samples among all studied returned
at least one
PCR-positive result for
C. pneumoniae (sample
509
AD, one of seven; sample 526
AD, two of five;
and sample 462
C, one of six [where the superscripts AD and
C indicate samples
from AD patients and controls, respectively]),
confirmation in
each instance was not observed in subsequent reactions
and these
samples were not considered truly positive according to the
above
criteria.
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FIG. 3.
PCR results. Example of PCR results for six hippocampal
DNA samples. (A) First round of amplifications with outer primer pair;
(B) second round of amplifications using the nested primer. A 100-bp
ladder was run (lanes labeled 100) alongside PCR products for size
determination.
|
|
Culture of PCR-positive samples.
A positive PCR result
together with the ability to culture C. pneumoniae from
clinical samples is considered a "gold standard" for its
identification. We attempted to culture C. pneumoniae from
our three PCR-positive samples along with PCR-negative samples from
congruent control regions. Following two passages, we were unable to
detect C. pneumoniae by PCR in either PCR-positive or -negative cultures. Control cultures of brain tissues seeded with C. pneumoniae during processing for culture consistently
gave positive cultures.
| |
CONCLUSION |
Based on the results of this study, we conclude that C. pneumoniae is neither strongly nor uniquely associated with the
neuropathology seen in late-onset AD. After a thorough examination of
AD and control brains, employing previously described methods and
confirmed in personal communications with the senior author of the
first and only report, C. pneumoniae could not be
unequivocally detected by PCR or culture methods in any CNS region from
AD cases or controls that we assayed. This observation differs
significantly from the original study, which claimed that C. pneumoniae DNA was extremely common in areas of AD neuropathology,
being detectable by PCR in 90% (17 of 19) of AD brains compared to 5%
(1 of 19) of control brains.
Notwithstanding the questionable detection of C. pneumoniae
DNA in three samples, the difference between the two sets of specimens is even more striking considering both the number of brain areas affected and the apparent levels of C. pneumoniae DNA
present in the PCR-positive specimens. It was reported previously that C. pneumoniae was detected in more than one CNS location in
brain tissue from 8 of the 17 AD cases (2). In this study
samples were considered positive if PCR results were positive in all
aliquots tested.
In the present study, every effort was made to use the same conditions
and methods as originally reported and subsequently modified. It is
unlikely that the differences in findings are attributable to technical
differences. Inadequate sample size also does not appear to be an
issue, given both the high frequency of PCR-positive samples reported
and the exhaustive testing of specimens in our study. This leaves
characteristics of the patient populations as a possible source of the
discrepancy. Of particular significance in this regard would be the
season of death as well as the institutional history of each patient
prior to death, as both of these circumstances could possibly
contribute to differences in the recentness of exposure to C. pneumoniae. All of the tissue samples analyzed in the original
study were obtained from three tissue banks associated with hospitals
on the East Coast, while our samples were obtained from a single bank
located in Southern California. These are different geographical
regions possibly differing in institutional practices and social
support systems.
C. pneumoniae possesses many attributes that would make it
an attractive contributory factor to the chronic and progressively degenerative neuropathological patterns seen in AD. Multiple
respiratory infections with chronic sequelae are very common, with
seroprevalence (immunoglobulin G) increasing throughout life, reaching
more than 75% among the elderly (7). C. pneumoniae is an obligate intracellular bacterium that can infect
a wide range of cell types. C. pneumoniae can chronically
infect and activate monocytes, which could traffic across the
blood-brain barrier. It is a gram-negative bacterium and thus produces
endotoxin. In short, it is the type of organism that could both
directly and indirectly stimulate the kind of inflammation seen within
AD lesions. Numerous studies have demonstrated conclusively its
presence within atherosclerotic plaques and in the synovial tissue of
some patients with reactive arthritis following respiratory infection
(P. Saikku, presented at the Proceedings of the Ninth International
Symposium on Human Chlamydial Infections, 1998). Most recently,
C. pneumoniae was detected by PCR in the cerebrospinal fluid
of 96% of multiple sclerosis patients, while being detected in only
18% of controls (14). Although the latter observation
requires confirmation, what is common to all these diseases, as well as
AD, is a chronic inflammatory process in which activated macrophages
participate. Knowing when and where activation occurs and the types of
responses chronically infected and activated macrophages can make when
circulating will help establish the nature of any causal relationship
between the presence of C. pneumoniae within inflammatory
lesions and the diseases which these lesions define. During such
chronic processes, the original of at least some of these cells might
prove to be a site of infection within the lung. However, the almost
universal and unequivocal association of C. pneumoniae with
the neurodegenerative process occurring during the terminal phase of AD
reported previously (2) was not confirmed in the present study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Neurosciences, Beckman Research Institute at the City of Hope, 1450 East Duarte Rd., Duarte, CA 91010. Phone: (626) 359-8111, ext. 3622. Fax: (626) 301-8948. E-mail: rring{at}coh.org.
| |
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Journal of Clinical Microbiology, July 2000, p. 2591-2594, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
