Previous Article | Next Article 
Journal of Clinical Microbiology, July 2000, p. 2512-2515, Vol. 38, No. 7
Département de Microbiologie et
Immunologie, Université de
Montréal,1 Département de
Microbiologie Médicale et Infectiologie, Centre Hospitalier de
l'Université de Montréal,2
Department of Oncology, McGill
University,3 and Centre de Recherche
du Centre Hospitalier de l'Université de
Montréal,4 Montréal, Québec,
Canada
Received 29 November 1999/Returned for modification 11 March
2000/Accepted 13 April 2000
We assessed the quality of genital samples submitted for
Chlamydia trachomatis detection by PCR by a second PCR
assay for the presence of human Individuals with Chlamydia
trachomatis infection of the genitourinary tract are often
asymptomatic or experience only mild symptoms (3).
Widespread screening of sexually active individuals for C. trachomatis infection has been advocated by the Centers for
Disease Control and Prevention to identify infected individuals who
require treatment to help control this epidemic (5). In recent years, the diagnosis of genital C. trachomatis
infections has been greatly improved by the application of nucleic acid
amplification techniques. The sensitivities of classical diagnostic
methods for the detection of C. trachomatis infections at
best reach 80% (3). Various nucleic acid amplification test
formats (PCR, the ligase chain reaction, transcription-mediated
amplification, the Q-Beta replicase assay, and strand displacement
amplification) have reported sensitivities and specificities of greater
than 90 and 99%, respectively (3).
The ability to detect C. trachomatis by PCR can be impaired
by the presence in clinical samples of substances inhibitory to Taq DNA polymerase (13, 17, 18). Concerns that
the performance of PCR could also be altered by the poor quality of
specimens have been confirmed in two studies (11, 20).
Although some diagnostic tests such as the direct fluorescence assay
concurrently screen for the presence of C. trachomatis and
columnar epithelial cells (10, 14, 20), commercial nucleic
acid amplification methodologies do not directly provide information on
the quality of the samples collected. The study described here was undertaken to evaluate the quality of
genital specimens routinely submitted for detection of C. trachomatis by PCR. The proportion of samples that contained small
amounts of cellular DNA was assessed by amplification of Specimen collection and processing.
This study tested for
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
An Important Proportion of Genital Samples
Submitted for Chlamydia trachomatis Detection by PCR Contain
Small Amounts of Cellular DNA as Measured by
-Globin Gene
Amplification
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-globin DNA. Endocervical and
urethral samples were first tested by the COBAS AMPLICOR C. trachomatis assay (Roche Diagnostic Systems) with an internal
control and were then amplified for the presence of -globin DNA with
primers PC04 and GH20. Samples that contained inhibitors were retested after dilution 1:10. A total of 407 genital samples (311 endocervical swabs from 311 women and 96 urethral swabs from 95 men and 1 woman) collected over a 1-month period were evaluated. The internal control could not be amplified, despite dilution, from 3 of 23 samples that
were retested after dilution because of inhibition, leaving 404 samples
that could be analyzed by PCR. Eleven samples tested positive for
C. trachomatis. Thirty (7.4%) of the 404 samples were
negative for -globin. Twelve of the 23 undiluted samples that
contained inhibitors tested positive for -globin DNA. Amplification of -globin DNA in samples submitted for C. trachomatis
detection by the COBAS AMPLICOR C. trachomatis assay
demonstrated that an important proportion of the samples did not
contain cellular DNA. Assessment of the quality of the samples for PCR
analysis by -globin amplification is feasible but cannot replace use
of the internal control.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-Globin amplification has been
used to assess the quality of genital specimens submitted for C. trachomatis or viral detection (4, 6, 9).
-globin
DNA. This is the first study on the adequacy of samples for the
detection of C. trachomatis that combined an internal control (IC) and a -globin control. Our study is also the first evaluation on the quality of urethral samples submitted for PCR.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-globin DNA in endocervical and urethral specimens submitted for
routine detection of C. trachomatis by PCR. Clinicians were
not warned of the ongoing evaluation of the quality of samples
submitted for C. trachomatis detection. All samples were
transported to the clinical microbiology laboratory within 24 h of
collection at room temperature and were processed within 3 days of collection.
-Globin and C. trachomatis DNA amplification.
Samples were tested for the presence of C. trachomatis DNA
with the COBAS AMPLICOR C. trachomatis test reagents and
system (Roche Diagnostic Systems) according to the manufacturer's
recommendations (17, 19). Briefly, 50 µl of processed
specimen was added to 50 µl of a master mixture that contained all
reagents for PCR and an IC DNA that was used to monitor the inhibition
of amplification (16, 17). This mixture was amplified in the
COBAS AMPLICOR instrument by using the thermal cycling conditions
programmed into the system. After amplification, the C. trachomatis and IC amplicons were denatured, hybridized in
separate reactions to amplicon-specific oligonucleotide probes bound to
magnetic microparticles, and detected with avidin-peroxidase and
colorimetric substrate. The failure to detect the IC DNA in a C. trachomatis-negative sample indicated that inhibition of PCR had
occurred (16). Specimens with inhibitory activity were
retested after dilution 10-fold in CT/NG Specimen Diluent (13, 17,
18). The measures used to control for contamination have been
described in previous publications (6, 7).
-globin DNA detection was performed with each
processed specimen to control for DNA integrity and for the presence of
an adequate quantity of human DNA (2, 7). Fifty microliters
of processed specimen was added to 50 µl of a master mixture that
contained 50 mM KCl, 5 U of Amplitaq DNA polymerase (Roche Diagnostic
Systems), dATP, dCTP, dGTP, and dTTP at a concentration of 400 µM
each, and 50 pmol of each primer (primers PC04 and GH20). Negative and
positive controls were included in each PCR run. The positive control
was a cell lysate from 10,000 HeLa cells per reaction mixture.
Amplifications were performed in a TC 9600 thermal cycler (Perkin-Elmer
Cetus, Montréal, Quebec, Canada) for 30 cycles with the following
cycling parameters: 95°C for 1 min, 55°C for 1 min, and 72°C for
1 min. PCR products were separated by electrophoresis on a 2% ethidium
bromide-stained agarose gel and were visualized on a UV
transilluminator. Samples that generated a 268-bp band were considered
positive. Samples that were found to be negative by PCR for -globin
were retested once. Ten microliters of the PCR products from these
samples was also spotted onto a nylon membrane, and the products were
allowed to hybridize with radiolabeled probe PC03 under the conditions described previously (1, 2, 15). Samples that were found to
be negative by PCR for -globin and the IC were retested after dilution 10-fold in CT/NG Specimen Diluent.
Statistical analysis. The Z test for differences in proportions for independent samples was used for statistical analysis of results (8). A P value of <0.05 was considered statistically significant.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Four hundred seven genital samples submitted for detection of
C. trachomatis by PCR were evaluated for the presence of
-globin DNA. Those specimens from 406 individuals included 311 consecutive endocervical swabs from 311 women and 96 urethral samples
from 95 men and 1 woman. The results obtained for -globin, C. trachomatis, and the IC are summarized in Table
1. The -globin PCR assay generated the
expected 268-bp band in the presence of a lysate of 100 human
fibroblasts or more processed as described in the Materials and Methods
section (data not shown). PCR products from specimens that did not
yield the 268-bp band on agarose gels were tested by a dot blot assay
with a radiolabeled probe. All scored negative for -globin,
demonstrating the absence of cellular DNA in the lysate (data not
shown).
|
Overall, inhibitors of the PCR, as measured by amplification of the IC,
were present in 23 (5.9%) of 407 samples. The rate of inhibition of
the PCR for endocervical specimens was lower than that for urethral
samples (0 of 311 [0%] versus 23 of 96 [24%]; P < 0.001). PCR inhibitors were removed after dilution 10-fold for 20 (87.0%) of the 23 urethral samples that contained inhibitors. Of the
latter 23 urethral specimens, 12 (52.2%) and 19 (82.3%) samples were
positive for -globin by PCR before and after dilution 10-fold,
respectively (Table 1). The 10-fold dilution of one sample tested
positive for -globin DNA, although PCR inhibition was still
demonstrated as a result of the failure to detect the IC. Excluding the
three samples with inhibition despite dilution, 404 genital specimens
(311 endocervical and 93 urethral samples) could be analyzed by PCR.
The differences in the rates of positivity for -globin between
endocervical and urethral samples were not significant (290 [93.3%]
of 311 endocervical samples versus 84 [90.3%] of 93 urethral specimens; P = 0.457). Of the 404 samples that could be
evaluated for DNA content, 30 (7.4%; 95% confidence interval, 4.9 to
9.8%) samples did not contain enough cellular DNA to test positive for the presence of -globin either by gel electrophoresis or by dot blot
detection of PCR products. The endocervical samples with low cellular
DNA contents were not provided by the same clinicians (data not shown).
C. trachomatis was detected in 11 individuals, including 7 women and 4 men. Of the 11 C. trachomatis-positive samples
from these individuals, 9 (81.8%) were positive for -globin,
whereas only 2 (18.2%) were negative for -globin (P = 0.004). Excluding the samples with inhibitors, the prevalence of
C. trachomatis infection was similar for -globin-positive
and -globin-negative samples (9 of 375 [2.4%] versus 2 of 30 [6.7%]; P = 0.418). However, due to the small number
of C. trachomatis-positive and -globin-negative samples,
the power to detect a difference between prevalence rates as
significant as 4.3% reached only 0.53.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This study demonstrates that several factors impede the detection
of C. trachomatis in genital samples submitted for PCR. Nearly 8.1% of samples could not be analyzed by PCR because they contained a small quantity of cellular DNA (30 samples) or because they
contained inhibitors (3 samples). C. trachomatis is an
intracellular organism that infects the columnar epithelial cells of
the cervix. The presence of an adequate number of cells directly
affects the sensitivity of culture, direct fluorescence assay, and PCR
for the detection of C. trachomatis (10-12, 14,
20). Moreover, two studies have demonstrated that the prevalence
of C. trachomatis infection as measured by PCR is greater
when samples contain genital cells (10, 20). The small
number of C. trachomatis-positive and -globin-negative
individuals in our study did not provide us with enough power to
evaluate the impact of the quality of the specimen on the rate of
detection of C. trachomatis.
Two studies support our finding that samples can test positive for C. trachomatis (11, 14, 20) even in the absence of cells or cellular DNA, possibly due to the presence of extracellular organisms. The absence of cells or cellular DNA indicates a reduced likelihood of C. trachomatis detection.
The quality of the specimen varies with the training and experience of
the individual who collects the samples (10). In several
studies, from 20 to 49% of samples submitted for C. trachomatis detection were inadequate on direct examination
(3, 10-12, 14, 20). The optimal number of cells required to
consider a sample adequate for PCR analysis and the percentage of
infected columnar cells in patients with asymptomatic C. trachomatis infection have not been established. In the future, a
comparison between the -globin PCR assay and Papanicolaou staining
of dry swabs could allow the establishment of a positivity threshold
for -globin DNA detection for the assessment of specimen quality.
However, the -globin-negative samples in this study did not contain
cellular DNA, as demonstrated by the absence of reactivity between PCR products and a radiolabeled probe. One limitation of the -globin PCR
is the lack of discrimination of the cell types in samples. Our rate of
inadequate specimens could thus be greater than 7%. Monitoring of
specimen adequacy and targeted training can have an impact on the
specimen adequacy rate (10, 12). We could not identify a
subset of clinicians who specifically collected improper genital specimens.
A single control that would allow screening for the presence of PCR
inhibitors and the presence of an adequate quantity of cellular DNA
would be interesting. However, -globin amplification cannot replace
IC amplification, since more than half of the urethral samples with
inhibitors scored positive for -globin. This discrepancy could be
explained by a larger number of target -globin DNA molecules compared with the small number of copies of the IC (13).
Direct smears cannot be performed with specimens contained in the
detergent-based transport media used for PCR. To avoid cell lysis, the
swab could be rolled onto a slide before inoculation in the transport
tube. However, most cells could be deposited on the staining slide and
would be lost for C. trachomatis analysis (20). A
second swab could be dedicated solely to the direct smear, but then the
actual quantity of cells introduced in the PCR mixture is unknown.
Rolling of the swab on the slide after inoculation of the transport
tube may result in the loss of cells for the evaluation of sample
adequacy. In one study, endocervical cells from dry swabs were
resuspended in 0.9% saline and an aliquot of the cell suspension was
stained with Papanicolaou stain. Such a protocol does not result in
cell loss for PCR but cannot be applied to specimens contained in PCR
transport medium (12). Cytological examination does not
control for the integrity of the DNA introduced into the amplification
reaction (11), while -globin detection evaluates the
integrity of the DNA directly in the lysate tested for C. trachomatis. The -globin PCR assay also allows one to test
samples in batches and does not require expertise in cytology.
-Globin amplification has been used to assess the quality of genital
specimens submitted for C. trachomatis or virus detection
(4, 6, 9).
The rate of false-negative results for C. trachomatis by PCR
can be diminished by screening for the presence of PCR inhibitors and
by treating samples to inactivate inhibitory substances (11, 13,
16, 17). Sample dilution (13, 17, 18) removed the PCR
inhibitors from most of our samples. Nearly all clinical specimens (19 of 23 samples; Table 1) still contained enough cellular DNA to generate
a positive -globin PCR result, despite their dilution 10-fold. PCR
inhibition has been reported in 7 to 19% (17, 18) of
endocervical swab specimens and in up to 45% of urethral swab
specimens (17). The lower inhibition rate obtained in this
study with endocervical swabs is related to the use of dry swabs that
are more sensitive than specimens contained in the Specimen Transport
Medium (11).
The Centers for Disease Control and Prevention recommends monitoring of
the quality of samples submitted for C. trachomatis detection (3, 5). The extent to which such a policy is
applied when nucleic amplification tests are used is unknown. Periodic testing for -globin in samples submitted for C. trachomatis detection could allow one to screen for improper
sampling techniques by clinicians and provide a tool for the assessment
of the quality of samples for PCR analysis but cannot replace the use
of the IC. Eventually, commercialized PCR tests could incorporate a
control for establishment of the integrity of DNA and for the presence of an adequate number of genital cells. Further studies should include
first-void urine samples, since these specimens are easier to collect
and may ensure a higher probability of assurance of sample adequacy.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Département de Microbiologie Médicale et Infectiologie, Hôpital Notre-Dame du Centre Hospitalier de l'Université de Montréal, 1560 Sherbrooke est, Montréal, Québec, Canada H2L 4M1. Phone: 514-281-6000, ext. 5103. Fax: 514-896-4607. E-mail: francois.coutlee{at}ssss.gouv.qc.ca.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Bauer, H. M., C. E. Greer, and M. M. Manos. 1992. Determination of genital human papillomavirus infection by consensus polymerase chain reaction amplification, p. 131-152. In C. S. Herrington, and J. O. D. McGee (ed.), Diagnostic molecular pathology, a practical approach. IRL Press, Oxford, United Kingdom. |
| 2. |
Bauer, H. M.,
Y. Ting,
C. E. Greer,
J. C. Chambers,
C. J. Tashiro,
J. Chimera,
A. Reingold, and M. M. Manos.
1991.
Genital human papillomavirus infection in female university students as determined by a PCR-based method.
JAMA
265:472-477 |
| 3. | Black, C. M. 1997. Current methods of laboratory diagnosis of Chlamydia trachomatis infections. Clin. Microbiol. Rev. 10:160-184[Abstract]. |
| 4. | Bobo, L. 1993. PCR detection of Chlamydia trachomatis, p. 235-241. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology. American Society for Microbiology, Washington, D.C. |
| 5. | Centers for Disease Control and Prevention. 1993. Recommendations for the prevention and management of Chlamydia trachomatis infections. Morb. Mortal. Wkly. Rep. 42:1-39[Medline]. |
| 6. |
Coutlée, F.,
P. Gravitt,
H. Richardson,
C. Hankins,
E. Franco,
N. Lapointe,
H. Voyer, and The Canadian Women's HIV Study Group.
1999.
Nonisotopic detection and typing of human papillomavirus DNA in genital samples by the line blot assay.
J. Clin. Microbiol.
37:1852-1857 |
| 7. | Coutlée, F., C. Hankins, N. Lapointe, and The Canadian Women's HIV Study Group. 1997. Comparison betwen vaginal tampon and cervicovaginal lavage specimen collection for detection of human papillomavirus DNA by the polymerase chain reaction. J. Med. Virol. 51:42-47[CrossRef][Medline]. |
| 8. | Fleiss, J. L. 1981. Statistical methods for rates and proportions. John Wiley & Sons Inc., New York, N.Y. |
| 9. |
Gravitt, P.,
C. L. Peyton,
R. J. Apple, and C. Wheeler.
1998.
Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by single-hybridization, reverse line blot detection method.
J. Clin. Microbiol.
36:3020-3027 |
| 10. | Kellogg, J. A. 1995. Impact of variation in endocervical specimen collection and testing techniques on frequency of false-positive and false-negative Chlamydia detection results. Am. J. Clin. Pathol. 104:554-559[Medline]. |
| 11. | Kellogg, J. A., J. W. Seiple, J. L. Klinedinst, E. S. Stroll, and S. H. Cavanaugh. 1995. Improved PCR detection of Chlamydia trachomatis by using an altered method of specimen transport and high-quality endocervical specimens. J. Clin. Microbiol. 33:2765-2767[Abstract]. |
| 12. | Kellogg, J. A., J. W. Seiple, C. L. Murray, and J. S. Levisky. 1990. Effect of endocervical specimen quality on detection of Chlamydia trachomatis and on the incidence of false-positive results with the Chlamydiazyme method. J. Clin. Microbiol. 28:1113. |
| 13. |
Mahony, J. B.,
S. Chong,
D. Jang,
K. E. Luinstra,
M. Faught,
D. Dalby,
J. Sellors, and M. A. Chernesky.
1998.
Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity.
J. Clin. Microbiol.
36:3122-3126 |
| 14. | Phillips, R. S., P. A. Hanff, R. S. Kaufman, and M. D. Aronson. 1987. Use of a direct fluorescent antibody test for detecting Chlamydia trachomatis cervical infection in women seeking routine gynecologic care. J. Infect. Dis. 156:575-581[Medline]. |
| 15. |
Resnick, R. M.,
M. T. E. Cornelissen,
D. K. Wright,
G. H. Eichinger,
H. S. Fox,
J. T. Schegget, and M. M. Manos.
1990.
Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers.
J. Natl. Cancer Inst.
82:1477-1484 |
| 16. | Rosenstrauss, M., Z. Wang, S.-Y. Chang, D. Debonville, and J. P. Spadoro. 1998. An internal control for routine diagnostic PCR: design properties and effect on clinical performance. J. Clin. Microbiol. 36:197. |
| 17. | Toye, B., W. Woods, M. Bobrowska, and K. Ramotar. 1998. Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis testing. J. Clin. Microbiol. 36:2358. |
| 18. | Verkooyen, R. P., A. Luijendijk, W. M. Huisman, W. H. F. Goessens, J. A. J. W. Kluytmans, J. H. van Rijsoort-Vos, and H. A. Verburgh. 1996. Detection of PCR inhibitors in cervical specimens by using the Amplicor Chlamydia trachomatis assay. J. Clin. Microbiol. 34:3072-3074[Abstract]. |
| 19. |
Vincelette, J.,
J. Schirm,
M. Bogard,
A. M. Bourgault,
D. S. Luijt,
A. Bianchi,
P. C. V. Vader,
A. Butcher, and M. Rosenstraus.
1999.
Multicenter evaluation of the fully automated COBAS AMPLICOR PCR test for detection of Chlamydia trachomatis in urogenital specimens.
J. Clin. Microbiol.
37:74-80 |
| 20. | Welsh, L. E., T. C. Quinn, and C. Gaydos. 1997. Influence of endocervical specimen adequacy on PCR and direct fluorescent-antibody staining for detection of Chlamydia trachomatis infections. J. Clin. Microbiol. 35:3078-3081[Abstract]. |
Journal of Clinical Microbiology, July 2000, p. 2512-2515, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Coutlee, F., Rouleau, D., Petignat, P., Ghattas, G., Kornegay, J. R., Schlag, P., Boyle, S., Hankins, C., Vezina, S., Cote, P., Macleod, J., Voyer, H., Forest, P., Walmsley, S., The Canadian Women's HIV Study Group, , Franco, E.
(2006). Enhanced Detection and Typing of Human Papillomavirus (HPV) DNA in Anogenital Samples with PGMY Primers and the Linear Array HPV Genotyping Test.. J. Clin. Microbiol.
44: 1998-2006
[Abstract] [Full Text] -
Chernesky, M., Castriciano, S., Jang, D., Smieja, M.
(2006). Use of Flocked Swabs and a Universal Transport Medium To Enhance Molecular Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.. J. Clin. Microbiol.
44: 1084-1086
[Abstract] [Full Text] -
Castriciano, S., Luinstra, K., Jang, D., Patel, J., Mahony, J., Kapala, J., Chernesky, M.
(2002). Accuracy of Results Obtained by Performing a Second Ligase Chain Reaction Assay and PCR Analysis on Urine Samples with Positive or Near-Cutoff Results in the LCx Test for Chlamydia trachomatis. J. Clin. Microbiol.
40: 2632-2634
[Abstract] [Full Text] -
Alary, M., Poulin, C., Bouchard, C., Fortier, M., Murray, G., Gingras, S., Aube, M., Morin, C.
(2001). Evaluation of a Modified Sanitary Napkin as a Sample Self-Collection Device for the Detection of Genital Chlamydial Infection in Women. J. Clin. Microbiol.
39: 2508-2512
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»