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Journal of Clinical Microbiology, May 2001, p. 1751-1756, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1751-1756.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Chlamydia trachomatis and
Neisseria gonorrhoeae by Enzyme Immunoassay, Culture, and
Three Nucleic Acid Amplification Tests
E.
Van Dyck,1,*
M.
Ieven,2
S.
Pattyn,1
L.
Van
Damme,1 and
M.
Laga1
STD/HIV Research and Intervention Unit,
Department of Microbiology, Institute of Tropical
Medicine,1 and Department of
Microbiology, University Hospital,2 Antwerp,
Belgium
Received 3 November 2000/Returned for modification 16 January
2001/Accepted 6 February 2001
 |
ABSTRACT |
The purpose of this study was to evaluate and compare three
commercially available nucleic acid amplification tests (NAATs) for the
detection of Neisseria gonorrhoeae and Chlamydia
trachomatis. Roche PCR and Becton Dickinson strand displacement
amplification (SDA) were performed on 733 endocervical swab specimens
from commercial sex workers. Abbott ligase chain reaction (LCR) was
performed on a subset of 396 samples. Endocervical specimens from all
women were also tested by culture for N. gonorrhoeae and by
Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis.
A positive N. gonorrhoeae result was defined as a positive
result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests.
According to these definitions, the sensitivities and specificities for
the subsample of 396 specimens of N. gonorrhoeae culture,
PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of
C. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The
performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by
discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities
of N. gonorrhoeae culture, PCR, and SDA before and after
LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%,
respectively. The sensitivities of C. trachomatis EIA, PCR,
and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%,
respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The
accuracies of the different NAATs were quite similar. SDA was the only
amplification assay with 100% specificity for detection of both
N. gonorrhoeae and C. trachomatis in
endocervical specimens.
| |
INTRODUCTION |
Chlamydia trachomatis and
Neisseria gonorrhoeae are two of the most prevalent sexually
transmitted pathogens, with high rates of infection among female
commercial sex workers in developing countries, a substantial
proportion of whom have asymptomatic infections. Traditional laboratory
diagnosis of these infections is done by culture for N. gonorrhoeae and cell culture or antigen detection for C. trachomatis. Recently, nucleic acid amplification tests (NAATs)
have become widely used; these tests have shown a greater sensitivity
and have improved the ability to detect C. trachomatis and
N. gonorrhoeae infections. Several studies have shown that
NAATs are more accurate than the former standard tests for C. trachomatis and N. gonorrhoeae (2-6, 11-13, 16, 22, 25-28, 30, 33). However, it is known that certain
substances in clinical specimens may be associated with amplification
inhibition and that NAATs may give false-positive results (7, 9,
16, 20, 21, 29, 31). Currently available commercial C. trachomatis and N. gonorrhoeae DNA amplification tests
include PCR (Roche Molecular Systems, Branchburg, N.J.), the ligase
chain reaction (LCR; Abbott Laboratories, Abbott Park, Ill.), and
strand displacement amplification (SDA; Becton Dickinson, Sparks, Md.).
A major drawback for comparative studies of different commercialized
NAATs for the detection of N. gonorrhoeae and C. trachomatis in genital swabs is the incompatibility of the various
specimen collection and transport systems, each accompanied by
particular handling instructions and DNA extraction procedures. To
allow a scientifically valid comparison and a correct head-to-head
evaluation of different diagnostic assays, all tests should be
performed on the same single specimen and the study should include a
substantial number of true-positive specimens.
The purpose of this study was to evaluate the performance of SDA, PCR,
and LCR in a reference laboratory setting for the detection of N. gonorrhoeae and C. trachomatis in single endocervical
swabs stored and transported in dry tubes. Locally performed N. gonorrhoeae culture and C. trachomatis enzyme
immunoassay (EIA), used for management of patients, were also evaluated.
| |
MATERIALS AND METHODS |
Study population and clinical specimens.
Between September
1996 and April 2000, a multicenter study on the effectiveness of a
vaginal microbicide to prevent human immunodeficiency virus (HIV)
infection among female commercial sex workers was conducted in Cotonou,
Benin; Durban, South Africa; and Hat Yai, Thailand. After giving
written informed consent, women were screened for HIV infection and
other sexually transmitted diseases, and HIV-negative women were
included in the study and followed up on a monthly basis. Dacron swabs
were used to collect endocervical specimens each month for the
detection of N. gonorrhoeae and C. trachomatis. A
first swab was used for N. gonorrhoeae culture on modified
Thayer Martin medium. A second swab was used for C. trachomatis EIA antigen detection (MicroTrak; Syva, San Jose, Calif.). A third swab was kept dry in a sterile cryovial at 4°C after
collection, stored at 
20°C within 5 h, and shipped on dry ice
to the Institute of Tropical Medicine, Antwerp, Belgium, for C. trachomatis-N. gonorrhoeae coamplification PCR testing (Amplicor; Roche Diagnostic Systems, Branchburg, N.J.). After arrival, swabs were
stored at 20°C until testing.
Sample preparation and processing.
Dry swabs (n = 733) collected consecutively between May 1999 and December 1999 (Cotonou, n = 317; Durban, n = 235; Hat
Yai, n = 181) were used for comparison of different
NAATs to detect N. gonorrhoeae and C. trachomatis. The first 396 specimens were tested in parallel by
PCR (Amplicor; Roche), SDA (BDProbeTec ET; Becton Dickinson), and LCR
(LCx; Abbott). The next 337 specimens were tested by PCR and SDA;
N. gonorrhoeae culture-negative, PCR-SDA-discordant samples
and samples positive by (C. trachomatis) EIA, PCR, or SDA
only were tested by LCR for N. gonorrhoeae or C. trachomatis, respectively.
For processing, specimens were thawed and kept at room temperature for
2 h, and 1.2 ml of diluted phosphate-buffered saline (9 parts
saline and 1 part phosphate-buffered saline) was added to each vial.
Samples were vortexed vigorously at maximum speed for 2 min. After
removal of the swabs, four aliquots of 250 µl of sample suspension
were pipetted in small conic tubes and used for immediate testing or
kept at 20°C for later testing.
For each amplification assay, a 250-µl sample was centrifuged at
12,000 ×
g for 10 min and the pellet was used for DNA
extraction,
as follows. (i) For PCR, 250 µl of Amplicor lysis buffer
was added
to the pellet. After vortexing for 30 s and incubation
for 15
min at room temperature, the sample was mixed with 250 µl of
specimen
diluent. After another vortexing, 50 µl of the treated
sample
was used for the PCR assay following the instructions of the
manufacturer.
(ii) For SDA, 1 ml of test-specific diluent was added to
the pellet.
After vortexing for 30 s, the manufacturer's
procedure was strictly
followed and 100 µl of the treated specimen
was used for amplification.
(iii) For LCR, 1 ml of test-specific urine
resuspension buffer
was added to the pellet. After vortexing for
30 s and heating,
100 µl of the treated specimen was used for
amplification according
to the instructions of the manufacturer. The
NAATs were performed
blindly by three different technicians. Amplicons
were detected
according to the different test procedures, and specimens
with
assay values equal to or greater than the cutoff were considered
positive.
Analysis of results.
Specimens tested in parallel by PCR,
SDA, and LCR were considered true positive for N. gonorrhoeae if they were positive by culture or by two NAATs and
true positive for C. trachomatis if they were positive by
any two tests (EIA, PCR, SDA, or LCR).
Specimens tested in parallel by PCR and SDA were considered
N. gonorrhoeae true positive if they were positive by culture
or by
both amplification assays and
C. trachomatis true positive
if they were positive by any two tests (EIA, PCR, or SDA). For
N. gonorrhoeae culture-negative, PCR-SDA-discordant samples and
for
C. trachomatis EIA-, PCR-, or SDA-only-positive samples,
N. gonorrhoeae LCR or
C. trachomatis LCR tests
were performed, and
LCR-positive samples were considered true positive
in discrepant
analysis.
The sensitivity and specificity of culture, PCR, SDA, and LCR for
N. gonorrhoeae and of EIA, PCR, SDA, and LCR for
C. trachomatis were calculated for specimens tested in parallel
(
n = 396) by
all three NAATs. The sensitivity and
specificity of
N. gonorrhoeae culture, PCR, and SDA and of
C. trachomatis EIA, PCR, and SDA
were also calculated for
all specimens (
n = 733) before and after
inclusion of
supplemental LCR data obtained for samples with discordant
results
after parallel testing. Ninety-five percent confidence
intervals (Cls)
were calculated based on the binomial distribution
of the observed test
results.
| |
RESULTS |
N. gonorrhoeae.
All 733 specimens were tested by
culture. The first 396 specimens were tested by PCR, SDA, and LCR in
parallel, the next 337 specimens were tested by PCR and SDA, and LCR
was performed on PCR-positive, SDA-negative, culture-negative samples
only. None of the samples was found to be inhibitory when tested for
the internal control in PCR or in SDA.
For the first 396 specimens, the pattern of test results is shown in
Table
1: 328 specimens were negative by
all tests, 63
(15.9%) specimens were
N. gonorrhoeae
positive, and 5 specimens
were positive in one test only. Table
2 shows the sensitivities
of culture,
PCR, SDA, and LCR. The 95% Cls revealed no differences
between the
three NAATs. Culture was significantly less sensitive
than PCR.
For the 337 samples tested by culture, PCR, and SDA, the results of all
three tests were identical for 310 (92.0%), while
55 (16.3%)
specimens were
N. gonorrhoeae culture positive or positive
by both PCR and SDA. Three culture-negative specimens were PCR
positive
but SDA negative. Table
2 shows the sensitivities of
culture, PCR, and
SDA. The 95% Cls revealed no differences between
the two NAATs.
Culture was significantly less
sensitive.
Table
3 shows the combined results of
culture, PCR, and SDA for all specimens (
n = 733).
According to the definitions, 115
samples were
N. gonorrhoeae positive: 78 (68.2%) cases were detected
by culture,
and 37 (32.2%) were detected by the combination of
PCR and SDA. In
addition, 8 samples were PCR positive but negative
by culture and by
SDA: 5 of these were LCR positive (3 were detected
among the first 396 samples, and 2 were detected among the 337
specimens, for which LCR was
performed on 3 discordant PCR-positive
samples only.
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TABLE 3.
Combined results of culture, PCR, and SDA for all 733 endocervical swabs tested for gonococcal infection
|
|
By combining all 733 specimens, the performance characteristics of
N. gonorrhoeae culture, PCR, and SDA can be estimated with
exclusion of all LCR data or with inclusion of the LCR results
for the
8 PCR-positive discordant samples. Sensitivities and specificities
of
culture, PCR, and SDA before and after discrepant analysis
by PCR are
shown in Table
4. Before discrepant
analysis the number
of
N. gonorrhoeae-positive samples was
115; sensitivities of culture,
PCR, and SDA were 67.8, 95.7, and
93.9%, respectively. After additional
testing by LCR of PCR-positive
discordant samples, the number
of
N. gonorrhoeae-positive
samples increased to 120 and the performance
of culture, PCR, and SDA
did not change significantly. Culture
was significantly less sensitive
than either NAAT.
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TABLE 4.
Performance characteristics of N. gonorrhoeae
culture, PCR, and SDA before and after resolution of discordant results
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|
C. trachomatis.
All specimens were tested by
enzyme-linked immunosorbent assay (ELISA). The first 396 specimens were
tested by PCR, SDA, and LCR, and the next 337 specimens were tested by
PCR and SDA, with LCR being performed on specimens showing one positive
result by either EIA, PCR, or SDA.
For the first 396 specimens, the pattern of test results is shown in
Table
5: 50 (12.6%) samples were
C. trachomatis positive,
and there were 7 PCR-only-positive
and 5 LCR-only-positive results.
Table
6
shows the sensitivities and specificities of ELISA, PCR,
SDA, and LCR.
ELISA was significantly less sensitive than the
NAATs.
For the 337 specimens tested by two NAATs (PCR and SDA), the results of
ELISA, PCR, and SDA were identical for 314 (93.2%),
while 24 (7.1%)
specimens were
C. trachomatis positive in at least
two
assays. Table
6 shows the sensitivities of ELISA, PCR, and
SDA. The
95% Cls revealed no differences between the two NAATs.
ELISA was
significantly less
sensitive.
The samples positive by one test only were additionally tested by LCR:
one ELISA-only-positive sample was positive by LCR,
and one out of
seven PCR-only-positive samples was positive by
LCR.
Table
7 shows the combined results of
ELISA, PCR, and SDA for the total of 733 specimens. According to the
definitions, 71
samples were
C. trachomatis positive; all 71 were identified by
both PCR and SDA, while ELISA identified 28 positive
samples.
One sample was positive by ELISA and negative by PCR and SDA
but
was positive by LCR; 16 samples were PCR positive and negative
by
ELISA and SDA, and 3 of these were LCR positive.
Because ELISA, PCR, and SDA were performed on all specimens,
sensitivities and specificities of the three tests can be estimated
with exclusion of all LCR results or with inclusion of the LCR
results
for ELISA-only-positive and PCR-only-positive samples.
The performance
characteristics of ELISA, PCR, and SDA before
and after discrepant
analysis by LCR are shown in Table
8.
Before
discrepant analysis, the number of
C. trachomatis-positive samples
was 71. After additional testing by
LCR of the samples positive
by one test only, the total number of
C. trachomatis-positive
samples increased to 75 and the
sensitivities of ELISA, PCR, and
SDA decreased slightly. The
specificity of SDA remained 100%;
for ELISA it changed from 99.8 to
100%, and for PCR it changed
from 97.6 to 98.0%.
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TABLE 8.
Performance characteristics of C. trachomatis
ELISA, PCR, and SDA with and without supplemental testing of discordant
results
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| |
DISCUSSION |
The performance and evaluation of NAATs for the detection of
N. gonorrhoeae and C. trachomatis in genital
specimens has been the subject of much study and controversy. We
compared the results of PCR, SDA, and LCR for N. gonorrhoeae
and C. trachomatis as well as those obtained by culture for
N. gonorrhoeae and by antigen ELISA for C. trachomatis.
The specificity of DNA amplification assays can be ensured by retesting
initially positive specimens by a different amplification method, as
was done in the present study. It is more difficult to evaluate the
sensitivity of NAATs (or any other assays) when they appear to be more
sensitive than the conventional reference test. Many investigators
repeat the test being evaluated on discordant specimens or subject them
to additional tests, a strategy known as discrepant analysis.
Discrepant analysis aims to identify, by an additional confirmatory
assay, true-positive samples, negative by the reference method but
positive by the test under evaluation. Since this procedure can improve
the apparent sensitivity and specificity of the new test, selective
supplemental testing favors the test being evaluated and introduces a
data bias (8, 10). The size of the bias will depend on the
sensitivity of the reference test and on the prevalence of disease. The
lower the sensitivity of the reference test is, the higher the increase
in specificity of the new test will be; the lower the prevalence of
disease, the higher the increase in sensitivity (8, 15,
18). An alternative approach is to use a combination of tests to
establish an expanded "gold standard" for the evaluation of a new
diagnostic test (1). The introduction of an additional
test to an expanded gold standard implies that it should be performed
on all the specimens. This approach, however, is not common practice
because it increases substantially the workload and the costs of a
study (17, 19, 24). In this study the results were
analyzed by applying an expanded gold standard and by discrepant analysis.
Commercial NAATs have their own specimen collection kits and transport
media; unfortunately, these are incompatible, creating a difficulty for
comparative studies. To overcome this inconvenience and to avoid
problems of specimen collection order and bias due to interswab
variation, we used single dry endocervical swabs. It has been shown
that specimens transported on dry swabs have a higher positivity rate
than swabs swirled for 15 s in transport medium and then discarded
(14).
Various levels of DNA amplification inhibition with clinical specimens
have been observed (20, 29, 31). Routine inclusion of an
internal control as provided by PCR and SDA (not by LCR) allows the
detection of amplification-inhibiting factors, validating the negative
results. No inhibition was observed in our samples.
In vitro culture is still the reference method for the diagnosis of
gonorrhea. The low sensitivity of N. gonorrhoeae culture (65%) found in our study is consistent with other publications reporting sensitivities ranging between 50 and 84% (2, 5, 6,
13). These data indicate that gonococcal infection in females as
defined by culture is significantly underdiagnosed. Reasons for
false-negative N. gonorrhoeae culture could include prior
antimicrobial therapy, loss of viability of the organisms during
transport, low concentrations of the organisms, or sampling error.
It has been shown in several recent studies that chlamydia culture,
previously considered to be the gold standard, has a sensitivity ranging from 50 to 85% in expert laboratories (4, 12,
23). Because of logistic problems and limited resources, we did
not perform C. trachomatis culture in this study but used
classic C. trachomatis antigen detection by ELISA for rapid
diagnosis and patient management. After confirmation of initially
positive samples by a blocking assay, the specificity of this test was 100%, but the sensitivity was extremely low (38.7%). Most studies performed on endocervical specimens found sensitivities ranging between
45 and 70% (16, 25, 26, 32). In a study performed by Toye
et al., 19 cases of C. trachomatis were detected by PCR, 8 were detected by culture, and none were detected by ELISA
(29). In our study there was a wide variation of the ELISA
sensitivity observed in the three participating centers, ranging from
12.8 to 63.6 to 71.4%. This variation most likely reflects a
combination of differences in skills between the clinicians collecting
the specimens, differences in transport and storage conditions, and variability in laboratory expertise.
In this study the performance of PCR, SDA, and LCR for the detection of
N. gonorrhoeae and C. trachomatis infection was
evaluated in 396 endocervical specimens, applying an expanded gold
standard. No significant difference was observed between the
sensitivities of PCR, SDA, and LCR, which were 95.2, 88.9, and 88.9%,
respectively, for N. gonorrhoeae and 98, 94, and 90%,
respectively, for C. trachomatis. For the detection of
N. gonorrhoeae the specificities of the NAATs were more than
99% and statistically similar. For C. trachomatis, the
specificities of PCR and LCR were 98 and 98.6%, respectively, versus
100% for SDA; this difference was not significant.
For a second series of 337 samples PCR and SDA were performed in
parallel, but LCR was done only on samples with discordant results for
N. gonorrhoeae and C. trachomatis. By combining
both series of samples, the performance characteristics of PCR and SDA
on 733 samples were compared before and after resolution of discrepant
results by LCR. For N. gonorrhoeae the initial sensitivities of PCR and SDA did not change significantly after resolution of discrepant results by LCR.
For C. trachomatis both PCR and SDA were 100% sensitive.
After resolution of the PCR-SDA-discrepant results and one
ELISA-positive, PCR-negative, SDA-negative result by LCR, the
sensitivity of PCR decreased slightly to 98.7% and the sensitivity of
SDA decreased significantly to 94.7%. After discrepant analysis the
specificity of PCR increased slightly and that of SDA remained unchanged.
The disparities between the sensitivities of the three NAATs used in
the present study may partly be explained by slight inoculum differences resulting from splitting of samples containing very low
numbers of organisms; the use of multiple swab specimens, however,
would probably have resulted in more disparities.
Although our results clearly show that sensitivity and specificity
estimates for NAATs may vary slightly or significantly depending on the
definition of the gold standard, it seems that all three assays are
well suited to screening for genital gonorrhea and chlamydial infection
in female endocervical specimens. SDA was the most accurate test in
this study, being 100% specific for both N. gonorrhoeae and
C. trachomatis.
| |
ACKNOWLEDGMENTS |
This study was part of a multicenter trial on vaginal
microbicides (Nonoxynol-9, COL 1492) funded by UNAIDS.
We thank Becton Dickinson for contributing diagnostic reagents. We are
grateful to Karin Janssens for her excellent secretarial assistance. We
also thank Hilde Smet and Vicky Cuylaerts for the laboratory testing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: STD/HIV Research
and Intervention Unit, Department of Microbiology, Institute of
Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium. Phone: 32 3 247 63 29. Fax: 32 3 247 63 33. E-mail:
evandyck{at}itg.be.
| |
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Journal of Clinical Microbiology, May 2001, p. 1751-1756, Vol. 39, No. 5
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.5.1751-1756.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
