![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Clinical Microbiology, February 2001, p. 519-524, Vol. 39, No. 2
Department of Clinical
Microbiology,1 and Department of
Infection Immunology,10 Hygiene-Institute, and
Department of Occupational and Social Hygiene, Institute of
Environmental Health,5 University of Vienna,
Vienna, Austria; Institute of Respiratory Diseases, IRCCS
Policlinico, University of Milan, Milan, Italy2;
Department of Clinical Virology, Umeå University Hospital, Umeå,
Sweden3; Division of Infectious Diseases,
Department of Medicine, The Johns Hopkins University, Baltimore,
Maryland4; Institute of Medical
Microbiology, Medical University of Lübeck, Lübeck,
Germany6; Research Laboratory for
Infectious Diseases, National Institute of Public Health and the
Environment, Bilthoven, The Netherlands7;
Department of Clinical Virology, Malmö General
Hospital, University of Lund, Lund,
Sweden8; and Department of Medical
Microbiology and Genitourinary Medicine, University of Liverpool,
Liverpool, United Kingdom9
Received 16 August 2000/Returned for modification 11 November
2000/Accepted 25 November 2000
The reported rate of detection of Chlamydia pneumoniae
DNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5)
were analyzed by 16 test methods in nine centers by means of PCR. The
positive controls were correctly identified to levels of 1, 0.1, and
0.01 inclusion bodies of C. pneumoniae/ml of tissue
homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods,
respectively. Three out of 16 negative controls (19%) were rated
positive. Positivity rates for atheroma samples varied between 0 and
60% for the different test methods, with the maximum concordant result
for positivity being only 25% for one carotid artery sample. There was
no consistent pattern of positive results among the various
laboratories, and there was no correlation between the detection rates
and the sensitivity of the assay used.
Accumulating evidence has been
supporting a role of Chlamydia pneumoniae, an
important respiratory pathogen in humans (9), in the
pathogenesis of atherosclerosis. Seroepidemiological studies suggesting
an association between C. pneumoniae and coronary
artery disease (18) were followed by the direct
demonstration of C. pneumoniae in the affected tissue by
means of PCR (1, 8, 10, 11, 15, 16, 19), which was found
to be more sensitive than cell culturing for this purpose.
PCR has the potential to detect a single organism, but its application
to clinical specimens must be considered with caution for a number of
reasons (2, 4). PCR results can vary widely; for the
detection of C. pneumoniae in atheromatous lesions, some study groups reported positivity rates of up to 100%, while others reported that PCR completely failed to detect the organism (7, 22).
To date, there are no standardized PCR or other nucleic acid
amplification methods for the detection of C. pneumoniae.
Current PCRs are all in-house assays using different primers, reaction conditions, and methods of detection. Furthermore, DNA extraction procedures prior to PCR vary between different laboratories. Apart from
differences in methodology, comparison of data proves difficult because
of the different clinical materials analyzed within individual studies.
Hence, it remains to be clarified whether these discrepancies in
positivity rates for C. pneumoniae in atheromas are the
result of methodological differences or whether they actually reflect differences in the amounts of C. pneumoniae in the materials
studied. The aim of this study was to compare established DNA
extraction methods and PCR protocols used by different centers by using
specimens as identical as possible.
Study design.
Nine laboratories with experience in the
detection of C. pneumoniae in atheromas by PCR received
panels consisting of aliquots of 20 samples identical in composition
but numbered by a participant-specific code. Each laboratory was free
to choose the preferred technique of DNA extraction and PCR.
Experimental panels.
Informed consent was obtained from
patients, and all procedures were done in accordance with institutional
human experimentation guidelines. Specimens (n = 15)
obtained from patients suffering from severe atherosclerosis and
therefore undergoing vascular surgery (carotid artery, n = 6; femoral artery, n = 1; aneurysm of the
abdominal aorta, n = 5; aortic valve, n = 2; and coronary artery, n = 1) were directly
transferred from the operating theater to the laboratory and frozen
immediately at
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.519-524.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Multicenter Comparison Trial of DNA Extraction
Methods and PCR Assays for Detection of Chlamydia
pneumoniae in Endarterectomy Specimens
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C. All clinical samples were thawed in one
session, decalcified and homogenized to 30 mg/ml in Dulbecco's
phosphate-buffered saline (PBS; Gibco Life Technologies, Paisley,
Scotland) using sterile tissue grinders, vortexed, immediately divided
into 2-ml aliquots, and refrozen at 80°C. Manipulations of negative
controls (fresh porcine aortic tissue, homogenized to 30 mg/ml in
Dulbecco's PBS) and surgical specimens were done in a vertical laminar
flow cabinet. Both the laboratory and the pipettes had never been used
for C. pneumoniae-related work before.
80°C until they were shipped on
dry ice by air freight to collaborating laboratories within 48 h.
Participants were requested to keep samples at 80°C until analysis
was performed.
DNA extraction methods and PCR assays.
Each laboratory
followed at least one of their established procedures for C. pneumoniae detection in atheromas by PCR. Table 1 displays
applied laboratory methodologies and summarizes the key variables of
the different testing methods.
|
Statistical analysis.
A mathematical model was applied based
on the straightforward assumption that the probability of a positive
reaction (Pr) depends only on the amount of DNA present in the specimen
and the sensitivity of the method to detect it. The only model which allows for a comparison of methods which is stochastically independent of the specific selection of tissue samples is the two-parametric logistic model:
![<UP>Pr</UP>(a<SUB>ij</SUB> = 1 ‖ &ggr;<SUB>i</SUB>,&sfgr;<SUB>j</SUB>) =<UP> exp</UP>(<UP>&ggr;</UP><SUB>i</SUB> − &sfgr;<SUB>j</SUB>)/[1 +<UP> exp</UP>(<UP>&ggr;</UP><SUB>i</SUB> − &sfgr;<SUB>j</SUB>)]](/content/vol39/issue2/fulltext/519/img001.gif)
|
i reflects the
amount of DNA present in tissue i, and 
j reflects the sensitivity of method
j. To simplify computations, the parameter
j is set proportional (factor 
) to the
detection limit or, more specifically, to its logarithm sj:
![<UP>Pr</UP>(a<SUB>ij</SUB> = 1 ‖ &ggr;<SUB>i</SUB>, s<SUB>j</SUB>,&agr;) =<UP> exp</UP>(<UP>&ggr;</UP><SUB>i</SUB> − &agr;s<SUB>j</SUB>)/[1 +<UP> exp</UP>(<UP>&ggr;</UP><SUB>i</SUB> − &agr;s<SUB>j</SUB>)]](/content/vol39/issue2/fulltext/519/img002.gif)
|
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Blinded, otherwise identical panels consisting of endarterectomy specimens and controls were analyzed in nine laboratories by 16 different PCR testing methods.
In three laboratories, the materials were analyzed more than once, with the same or different primers and with single-round or nested PCR. For analysis and discussion, each testing method was considered a separate experiment; therefore, each attempt was treated as a separate analysis.
The sample volume used for DNA extraction was in the range of 50 µl (1.5 mg) to 2 ml (60 mg) of tissue homogenate, and five different commercially available kits as well as standard phenol-chloroform DNA preparation methods were used. Three different species-specific gene regions of C. pneumoniae served as target sequences for PCR: the 16S rRNA gene (5 protocols), the omp-1 gene (10 protocols), and a cloned PstI fragment (1 protocol). PCR setups were as follows: single-round PCR, 5 protocols; nested PCR, 10 protocols; and real-time PCR, 1 protocol. Some laboratories performed a touchdown technique (nine protocols). Four different methods of PCR product detection were applied: agarose or polyacrylamide gel electrophoresis and ethidium bromide staining (eight protocols); hybridization techniques, such as Southern blotting with biotinylated probes (five protocols); detection by an enzyme-linked immunosorbent assay (two protocols); and real-time PCR (one protocol). None of the participants applied sequencing techniques. Table 1 displays and contrasts the 16 testing methods applied in this study.
Controls.
The results obtained from the blinded controls are
shown in Table 2. All 16 testing methods
identified the positive controls as positive to the level of one
C. pneumoniae inclusion or 500 chlamydial particles/ml of
tissue homogenate (100%). Furthermore, 11 testing methods (69%)
allowed the detection of 0.1 chlamydial inclusion (50 particles) and 3 protocols (19%) allowed the detection of 0.01 chlamydial inclusion (5 particles) per ml of tissue homogenate. Taking into account the amount
of tissue used for DNA preparation and finally the volume of DNA
extract used for PCR when calculating the detection limit for C. pneumoniae in spiked tissue samples, the achieved sensitivities
were in the range of 0.3 to 0.0001 inclusion, corresponding to 1.5 × 102 to 5 × 10
2 particles (Table
3). Three out of 16 negative controls
(19%) were rated positive.
|
|
Endarterectomy samples. In general, 240 analyses were performed on 15 atheroma samples by means of 16 testing methods. In summary, 21 out of the 240 analyses (9%) were considered positive, but none of them was rated positive by more than 4 out of 16 protocols, showing a maximum concordant result for positivity of only 25% for one carotid artery.
Out of 240 analyses performed on 15 samples, 219 (91%) were reported negative. Two laboratories (G [detection methods 1, 2, and 3] and H) spiked specimens with C. pneumoniae DNA and reported their DNA extracts to be free of PCR inhibitors. Of the 15 endarterectomy samples, 4 (27%) were rated negative by all 16 testing methods.Statistical analysis.
Based on the two-parameter logistic
model, the expected number of positive results for each method was
calculated (Table 4). A priori,
differences could be expected due to the differences in the
sensitivities of the methods. Considering sensitivity as determined by
the positive controls, a consistent variation of about 1:2 is to be
expected for low positivity and less variation is to be expected for
higher positivity. Apparently, there is no relationship between the
numbers of specimens reported positive and the sensitivities of the
methods.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Several studies evaluating PCR for the detection of C. pneumoniae in vessel wall specimens have been published in recent years (2). Only one study compared detection methods between different centers (17). In that study, coronary arteries from patients seeking heart transplantation were investigated. At least one testing method showed C. pneumoniae in coronary arteries in 7 of 10 patients with coronary atherosclerosis. The concordant positivity in that study ranged between 9 and 82%. Since different parts of the atherosclerotic vessels were analyzed in the participating laboratories, a random distribution of C. pneumoniae within the plaques may explain some of the discrepancies in the detection of this organism. However, lack of standardization of diagnostic methods also may be responsible for false-positive as well as false-negative results.
Our study was conducted to compare DNA extraction methods and PCR
protocols
as are established in different centersusing aliquots of
homogenized specimens. We are aware that such aliquots are similar but
not absolutely identical. The number of targets in clinical samples
might be very low and aliquots of the same sample may contain, as a
result of a random distribution, various numbers of C. pneumoniae DNA targets.
In our study, there was no consistent pattern of positive results among the various laboratories, and the rate of detection of positive results (0 to 60%) in the various laboratories did not correlate with the sensitivity of their assays, as determined with a panel of spiked control specimens. Some laboratories reported positive controls to 0.01 inclusion or five chlamydial particles/ml as positive but reported no positive results for the atheroma specimens; however, others reported several atheroma specimens positive but some of the positive controls negative. Therefore, neither an extraordinarily high sensitivity nor an accumulation of aliquots with a relatively high copy number (which is, displayed in Table 4, statistically extremely unlikely) could explain the high percentage of positive findings in laboratories A and B.
Contamination of negative controls occurred in two laboratories, but the methods affected were not the methods that provided the largest number of positive results for the atheroma specimens (in fact, one of them reported none of the atheroma specimens positive). Therefore, the observed contamination seems to be related to the high copy number of the positive control preceding the negative control. As suggested by Boman et al. (2), the use of strongly positive controls, i.e., highly concentrated solutions of extracted C. pneumoniae DNA, should be avoided in future studies.
A possible drawback of this study was the inclusion of only one negative control, which might not be enough to determine cross contamination. However, "in-house" negative controls were used by the individual laboratories (range, 2 to 10 in each run per testing method), and only results of testing attempts with correctly identified negative as well as positive controls were considered for analyses. Furthermore, taking into account that more than 90% of the atheroma samples were rated negative, contamination was not a major problem in this study. Additionally, it should be emphasized that the primary goal of this study was the comparison of already established protocols but not the determination of sensitivities and specificities of each method, a strategy which makes the number of control samples less critical.
The overall detection rate (9%) and the maximum concordant positive rate (25% for one carotid artery) for the atheroma samples were lower than expected. Possible explanations for these rather unexpected findings are DNA degradation due to grinding of the samples and the release of endonucleases (13) and the presence of PCR inhibitors within clinical samples. Two laboratories evaluated samples for inhibition and reported the absence of detectable PCR inhibitors. Although only sections of the vessel walls without obvious calcifications, thrombi, or fatty tissue were processed, one cannot conclude that all samples prepared by the remaining protocols were free of PCR inhibitors, since PCR inhibition was not studied by all participants. We tried to minimize the presence of PCR inhibitors and therefore removed the calcified stony parts of the vessel walls, which are known PCR inhibitors. If it is assumed that this part of a specimen contains C. pneumoniae, it must be taken into account that the specimens used in this study were from patients suffering from extensive and advanced atherosclerotic lesions. Therefore, it is very likely that C. pneumoniae, if present, would have disrupted the endothelial barrier and been distributed into surrounding areas of the vessel walls at the time of surgical intervention.
The results of this study indicate that procedures suitable for extracting DNA from spiked control samples are not necessarily suitable for extracting DNA from clinical samples. Hence, DNA extraction by itself could be a determining factor. Different DNA extraction protocols with minimized variables and performed simultaneously by participating laboratories, followed by "DNA sharing" and PCR analysis, could help to answer this question in future studies.
Although this study was not designed to find out whether or not C. pneumoniae is present in atheromatous tissues, the large number of negative results obtained with the majority of protocols remains difficult to explain. Unfortunately, during the study period, no quantitative amplification technique had been established and validated against a conventional PCR protocol in any of the participating centers. Such a quantitative PCR might have provided a better definition of the actual chlamydial load and a consensus regarding true-positive results in this study.
It can be concluded that C. pneumoniae is present only at a very low target level in atheromatous specimens, if at all; that current detection methods are relatively reliable; and that discordant positivity rates are generated as a result of a problem with the distribution of C. pneumoniae targets (i.e., due to sampling error). However, the statistical calculation concerning the observed distribution of positive results in the atheroma panel does not support these assumptions. Rates of detection of between 15 and 60% have been reported in previous studies (1, 7, 8, 10, 11, 15, 16, 19, 22). Based on an average positivity rate of 30 to 40%, as reported in the literature, the expected and observed distributions of positive results differ significantly (Table 4). If the only difference between methods is sensitivity, the probability of observing the distribution found in this study just by chance is less than 1 in a million!
It is impossible to decide which methods are correct or whether any of the methods applied is correct. It is possible that the clinical specimens analyzed contained almost no C. pneumoniae DNA, that the occasional positive results resulted from incidental contamination, and that the few methods yielding high proportion of positive results had an inherent yet undetected error. However, it is also possible that C. pneumoniae DNA was present in the majority of samples and that the few methods yielding positive results were closer to the truth, while those failing to yield a positive reaction could not detect the target either due to the failure of DNA extraction from atheroma tissues or due to some undetected inhibitory mechanism. Since no reference method is available, a decision between these two explanations is impossible to make.
| |
ACKNOWLEDGMENTS |
|---|
We thank Peter Polterauer for providing surgical specimens. We are indebted to the staff of Operating Theater Group II at Vienna University Hospital, whose personal engagement made the transport of specimens possible. We also appreciate Hermelinde Steiner's assistance in preparing the manuscript. Furthermore, the excellent technical assistance of Marion Loidl is gratefully acknowledged.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Clinical Microbiology, Hygiene-Institute, University of Vienna, Vienna University Hospital, Währinger Gürtel 18-20/5P, A-1090 Vienna, Austria. Phone: 43 (1) 40400-5151. Fax: 43 (1) 40400-5228. E-mail: Petra.Apfalter{at}akh-wien.ac.at.
Present address: Department of Infection, Virology Section, St.
Thomas' Hospital, London SE 1 7EH, United Kingdom.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Blasi, F., F. Denti, M. Erba, R. Cosentini, R. Raccanelli, A. Rinaldi, L. Fagetti, G. Esposito, U. Roberti, and L. Allegra. 1996. Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J. Clin. Microbiol. 34:2677-2769. |
| 2. |
Boman, J.,
C. A. Gaydos, and T. C. Quinn.
1999.
Molecular diagnosis of Chlamydia pneumoniae infection.
J. Clin. Microbiol.
37:3791-3799 |
| 3. |
Campbell, L. A.,
M. Perez Melgosa,
D. J. Hamilton,
C. C. Kuo, and J. T. Grayston.
1992.
Detection of Chlamydia pneumoniae by polymerase chain reaction.
J. Clin. Microbiol.
30:434-439 |
| 4. | Fredricks, D. N., and D. A. Relman. 1999. Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin. Infect. Dis. 29:475-488[Medline]. |
| 5. | Gaydos, C. A., C. L. Fowler, V. J. Gill, J. J. Eiden, and T. C. Quinn. 1993. Detection of Chlamydia pneumoniae by polymerase chain reaction-enzyme immunoassay in an immunocompromised population. Clin. Infect. Dis. 17:718-723[Medline]. |
| 6. |
Gaydos, C. A.,
T. C. Quinn, and J. J. Eiden.
1992.
Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.
J. Clin. Microbiol.
30:796-800 |
| 7. | Jantos, C. A., A. Nesseler, W. Waas, W. Baumgärtner, H. Tillmanns, and W. Haberbosch. 1999. Low prevalence of Chlamydia pneumoniae in atherectomy specimens from patients with coronary heart disease. Clin. Infect. Dis. 28:988-992[Medline]. |
| 8. | Juvonen, J., T. Juvonen, A. Laurila, H. Alakärppä, K. Lounatmaa, H. M. Surcel, M. Leinonen, M. I. Kairaluoma, and P. Saikku. 1997. Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms. J. Vasc. Surg. 25:499-505[CrossRef][Medline]. |
| 9. | Kuo, C. C., L. A. Jackson, L. A. Campbell, and J. T. Grayston. 1995. Chlamydia pneumoniae (TWAR). Clin. Microbiol. Rev. 8:451-461[Abstract]. |
| 10. | Kuo, C. C., A. Shor, L. A. Campbell, H. Fukushi, D. L. Patton, and J. T. Grayston. 1993. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J. Infect. Dis. 167:841-849[Medline]. |
| 11. | Maass, M., E. Krause, P. M. Engel, and S. Krüger. 1997. Endovascular presence of Chlamydia pneumoniae in patients with hemodynamically effective carotid artery stenosis. Angiology 48:699-706. |
| 12. |
Madico, G.,
T. C. Quinn,
J. Boman, and C. A. Gaydos.
2000.
Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes.
J. Clin. Microbiol.
38:1085-1093 |
| 13. |
Meijer, A.,
J. A. van der Vliet,
P. J. M. Roholl,
S. K. Gielis-Proper,
A. de Vries, and J. M. Ossewaarde.
1999.
Chlamydia pneumoniae in abdominal aortic aneurysmsabundance of membrane components in the absence of heat shock protein 60 and DNA.
Arterioscler. Thromb. Vasc. Biol.
19:2680-2686 |
| 14. | Meijer, A., J. A. van der Vliet, L. M. Schouls, A. de Vries, P. J. M. Roholl, and J. M. Ossewaarde. 1998. Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR in the absence of a gold standard. Res. Microbiol. 149:577-583[Medline]. |
| 15. | Nadrchal, R., A. Makristathis, P. Apfalter, M. Rotter, W. Trubel, I. Huk, P. Polterauer, and A. M. Hirschl. 1999. Detection of Chlamydia pneumoniae DNA in atheromatous tissues by polymerase chain reaction. Wien. Klin. Wochenschr. 111:153-156[Medline]. |
| 16. |
Ong, G.,
B. J. Thomas,
A. O. Mansfield,
B. R. Davidson, and D. Taylor-Robinson.
1996.
Detection and widespread distribution of Chlamydia pneumoniae in the vascular system and its possible implications.
J. Clin. Pathol.
49:102-106 |
| 17. |
Ramirez, J. A., and the Chlamydia pneumoniae/Atherosclerosis Study Group.
1996.
Isolation of Chlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis.
Ann. Intern. Med.
125:979-982 |
| 18. | Saikku, P., M. Leinonen, L. Tenkanen, E. Linnanmäki, M. R. Ekman, V. Manninen, M. Mänttäri, M. H. Frick, and J. K. Huttunen. 1992. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann. Intern. Med. 116:273-278. |
| 19. |
Thomas, M.,
Y. Wong,
D. Thomas,
M. Ajaz,
V. Tsang,
P. J. Gallaher, and M. E. Ward.
1999.
Relation between direct detection of Chlamydia pneumoniae DNA in human coronary arteries at postmortem examination and histological severity (Stary grading) of associated atherosclerotic plaque.
Circulation
99:2733-2736 |
| 20. | Tong, C. Y. W., C. Donnelly, G. Harvey, and M. Sillis. 1999. Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. J. Clin. Pathol. 52:257-263[Abstract]. |
| 21. |
Tong, C. Y. W., and M. Sillis.
1993.
Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.
J. Clin. Pathol.
46:313-317 |
| 22. | Weiss, S. M., P. M. Roblin, C. A. Gaydos, P. Cummings, D. L. Patton, N. Schulhoff, J. Shani, R. Frankel, K. Penney, T. C. Quinn, M. R. Hammerschlag, and J. Schachter. 1996. Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy. J. Infect. Dis. 173:957-962[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. |
|---|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
