Detection of Chlamydia trachomatis endocervical infection in asymptomatic and symptomatic women: Comparison of deoxribonucleic acid probe test with tissue culture

Detection of Chlamydia trachomatis endocervical infection in asymptomatic and symptomatic women: Comparison of deoxribonucleic acid probe test with tissue culture Lisa I. Yang, MD,. Elizabeth S. Panke, MD, PhD," Phyllis A. Leist, PhD,c Richard J. Fry, MD,. and Richard F. Lee, PhDb Cincinnati, Ohio A deoxyribonucleic acid probe assay (PACE 2, Gen-Probe, San Diego) was compared with a standard tissue culture method for detection of Chlamydia trachomatis endocervical infection in both asymptomatic and symptomatic women. The results of the probe test were expressed as a ratio of relative light units of the specimen per relative light units of the cutoff recommended by the manufacturer. Samples with sample/cutoff ratios near 1.0 were repeated until two or more consistent ratios were obtained. A total of 426 specimens were obtained, with an overall disease prevalence of 10.1 %. Of the 426 specimens examined, seven (1.6%) were near the cutoff and were retested. The results of 426 samples with matching cultures indicated that the manufacturer's discrete cutoff was adequate for results determination. The deoxyribonucleic acid probe test was essentially equivalent to standard tissue culture in terms of sensitivity, specificity, and positive and negative predictive values in a low-prevalence patient population. (AM J OBSTET GVNECOL 1991 ;165:1444-53.)

Key words: Chlamydia trachomatis, deoxyribonucleic acid probe, tissue culture Infection by Chlamydia trachomatis is recognized as the most prevalent sexually transmitted disease worldwide, with an estimated four million cases per year in the United States,I.2 While C. trachomatis is an important pathogen in lower and upper genital tract infections and pregnancy complications, a substantial number of cases occur without symptoms in patients.I.3·4 The most important factor in controlling the chlamydia epidemic is correct and timely diagnosis. This has previously been limited to cell culture, which is considered the gold standard in spite of < 100% sensitivity. New techniques include antigen detection with enzyme immunoassay, direct fluorescent antibody, and deoxyribonucleic acid (DNA) probe tests. PACE 2 (Cen-Probe, San Diego) is a commercially available, rapid detection test for C. trachomatis endocervical and urethral infection. The PACE 2 system uses the technique of nucleic acid hybridization to identify C. trachomatis DNA directly from urogenital swab specimens. Nucleic acid hybridization tests are based Oil the From the Department of Obstetrics and Gynecology: the Molecular Diagnostics Center,' and the Department of Pathology,' Good Samaritan Hospital. Supported in part by Education and Research Foundation, Good Samaritan Hospital, Cincinnati, Ohio. Received for publication November 2, 1990; revised March 20, 1991; accepted April 8, 1991. Reprint requests: Lisa I. Yang, MD, Department of Obstetrics and Gynecology. Good Samaritan Hospital. 3217 Clifton Ave.. Cincinnati. OH -/5220-2-/89. 6/1/30055

1444

ability of complementary nucleic acid strands to specifically align and form stable double-stranded complexes. PACE 2 uses a single-stranded DNA probe labeled with a chemiluminescent marker. The probe is complementary to ribosomal ribonucleic acid (rRNA) of the target organism. If rRNA from C. trachomatis is present in a collected specimen, the labeled DNA probe combines with it to form a stable DNA/RNA hybrid. Magnetic spheres are added to the reaction and these bind only to the stable hybrids. The labeled hybrids are magnetically separated from the nonhybridized probe and measured in a luminometer. A greater light response is generated with increasing numbers of stable hybrids present in the specimen (Fig. 1). The luminometer and magnetic separation unit are the only specialized laboratory equipment needed to perform this test. The reagents are commercially available from Cen-Probe. Other materials required include water bath, vortex mixer, micropipettes, and pipettes that are routinely available in most clinical laboratories. The purpose ofthis study was to compare a new DNA probe assay format, designed to increase sensitivity and simplify procedural steps, with a standard cell culture method for the detection of C. trachomatis in endocervical swab specimens in both asymptomatic and symptomatic women seen for obstetric and gynecologic care. Currently, chlamydia infection may remain undiagnosed, misdiagnosed, or empirically treated with antibiotics because the standard diagnostic test, namely, cell culture, remains unavailable in smaller hospitals, is

DNA probe test vs culture for C. trachomatis

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~

~ ~ Magnetic Separation

1445

\Vashed Unbound probe

Measure Luminescence

DNA-rRNA complexes

• - Chlamydia rRNA DNA probe Chemiluminescent marker

GJ - Single-stranded

--w -

Fig. 1. Nucleic acid hybridization test for the identification of C. tmcholl/ati:; DNA from urogenital swab specimens.

labor intensive, and requires a lengthy turnaround time. The development of a reliable, rapid, and easyto-use laboratory test for the detection of chlamydial infection has important ramifications for patients and clinicians. We also evaluated whether the discrete manufacturer-recommended cutoff value is sufficient for unequivocal reporting of results for all patient specimens. Methods

Patient population. Patients in the study were divided into two groups. Group 1 consisted of asymptomatic pregnant women who were seen for prenatal care. Patient charts were reviewed to determine the presence or absence of symptoms related to chlamydial infection at the time of specimen collection. Group 2 consisted of women seen for gynecologic care with symptoms of lower genital tract infection (i.e., vaginal discharge, pelvic pain, dyspareunia, coexisting sexually transmitted disease), patients requesting cultures because of known sexually transmitted disease exposure, and pregnant women with symptoms. Patients receiving antibiotics (duration, ~4 weeks) before specimen collecton and individuals with no clinical histories were excluded from the study. All patients were seen at Good Samaritan Hospital Outpatient Clinic or Group Health Associates, Cincinnati, between September 1989 and June 1990. Specimen collection. The exocervix was first cleansed of excess mucus with a Dacron swab. Subsequently, two endocervical specimens were obtained, one for DNA probe test and one for C. trachomatis culture. The samples were collected in no designated order, and

collection was not randomized. However, collection was done by 17 different physicians, thus avoiding systematic collection bias. Specimens collected for DNA probe test were placed in Gen-Probe transport tubes containing 1.0 ml of specimen preservative and glass beads. Samples were stored at room temperature for up to 1 week before being tested. For culture, specimens were placed in chlamydia transport media (2-sucrose-phosphate media, Bartel's Immunodiagnostic Supplies, Bellevue, Wash.) and processed immediately or stored at 4° C for no longer than 24 hours. DNA probe assay. The Gen-Probe PACE 2 system chemiluminescent-labeled DNA probe test was used in the study. This system uses an acridinium-ester-labeled single-stranded DNA probe that is complementary to ribosomal rRNA of C. trachomatis. 5 All reagents and samples were brought to room temperature before assay. Each sample was mixed in a vortex for 10 to 15 seconds. Swabs were then expressed of liquid on the side of the tube and discarded. Reconstituted probe reagent (100 J..Ll) was pipetted into each tube. Specimens were mixed in a vortex, and then 100 J..Ll of sample was added to the probe. The tubes were covered with a sealing card and the tube rack was shaken three to five times to mix. The mixture was incubated at 60° ± 1° C water bath for 1 hour. One milliliter of separation solution was added to each tube and then incubated at 60° ± 1°C for 10 minutes. The separation rack was placed onto a magnetic base at room temperature for 5 minutes. The supernatant was decanted and the tubes blotted on absorbent paper. Each tube was filled to the rim with wash solution. The tubes remained on the separation

1446 Yang et al.

rack for 20 minutes at room temperature The supernatant was then decanted but not blotted. The tube rack was shaken to resuspend the pellets. For each set of specimen runs, three negative controls and one positive control were simultaneously processed. The tubes were read on the Leader I luminometer (Cen-Probe) with the chlamydia protocol selected. The results of the test were calculated based on the difference between the response relative light units (RLU) of the specimen and the mean relative light units of the three negative references. Culture. Specimens were mixed in a vortex with sterile glass beads for 60 seconds, and debris was removed by centrifugation at 900 g for 5 minutes. Supernatant (200 I.d) was inoculated into each of two shell vials containing McCoy cell monolayers on coverslips (Bartel's Immunodiagnostic Supplies, Bellevue, Wash.). Vials were centrifuged for 1 hour at 3000 g at 25° C. Supernatant was aspirated, and 1.0 ml of chlamydia culture media with cycloheximide was added to the monolayers. Vials were incubated at 35° C for 48 hours. The coverslip from one vial was fixed in chilled acetone and stained with fluorescein-labeled antichlamydial mouse monoclonal antibody reagent (Bartel's). Coverslips were examined' at a magnification of x 100 and inclusions were confirmed at x 400 with an Olympus fluorescent microscope. A culture was considered positive if one or more inclusions were present. Passage of the second vial was performed on questionable one-vial results, monolayers showing elementary bodies but no inclusions, toxic reactions, or any specimen with discrepant probe and culture results. Discrepant results. All specimens were stored at - 70° C for further reference if discrepant results occurred. When discrepant results were obtained, specimens were submitted in blind fashion to Cen-Probe for analysis by probe competition assays. The samples were tested with the PACE 2 assay format with halfvolume reactions (50 Id sample and 50 I.d probe). Three separate assays were performed for each specimen: (1) sample and labeled C. trachomatis probe, (2) sample and labeled C. trachomatis probe and x 100 unlabeled homologous probe (C. trachomatis), and (3) sample and labeled C. trachomatis probe and x 100 unlabeled heterologous probe (Mycoplasma pneumoniae). A 90% reduction in difference expressed in relative light units (competition) in tube No.2 versus tubes No.1 and No. 3 indicated that a specific hybridization reaction took place and that C. trachomatis nucleic acids were present in the sample. A reduction <90% indicated that a specific hybridization reaction had not taken place (no competition) and that C. trachomatis nucleic acids were not present in the sample (Fig. 2). In addition, DNA probe assays were performed on all specimens submitted for culture when culture was positive and DNA probe assay was negative.

November 1991 Am J Obstet Gynecol

Cutoff range for the DNA probe assay. The results of the DNA probe test were calculated on the basis of the difference between the response in relative light units of the specimen and the mean of three negative reference values. According to the manufacturer, the specimen should be considered positive for C. trachomatis if the difference between the specimen response and the mean of the negative reference is ~300 RLU. One of the objectives of the study was to determine if using such a discrete manufacturer recommended cutoff value is sufficient for unequivocal reporting of results for all patient samples. This question is especially important for patient samples that may have values in relative light units that are close to the recommended cutoff value. Therefore we obtained interassay precision data for samples at various levels from the cutoff value, examined the distribution of values in relative light units for all patient samples in relation to the cutoff value, and correlated the DNA probe test results with those of tissue culture and clinical data. All patient values in relative light units were expressed as a ratio of the sample relative light units to the cutoff relative light units recommended by the manufacturer. Ratios ~ 1.0 represented patient specimens that were positive for C. trachomatis (i.e., difference ~300 RLU). Ratios < 1.0 represented patient specimens that were negative for C. trachomatis (i.e., difference <300 RLU). Additionally, more decisive negative or positive results were obtained the further a given ratio deviated from the value of 1.0. Specimens with ratios near 1.0 were multiply tested to determine if a cutoff range rather than a discrete cutoff point could be determined for more accurate results. Statistics. Nonparametric comparisons of categoric variables used X2 analysis. Calculations for test validity were made with standard formulas for sensitivity, specificity, and predictive values. Results

A total of 426 endocervical specimens were evaluated by tissue culture and by the Cen-Probe PACE 2 DNA probe assay. An additional 2703 patient specimens without matching cultures were analyzed by DNA probe only. These additional samples were used to obtain more precise analysis of borderline specimens and to verify the distribution of study samples around the cutoff. Interassay precision data. Table I shows betweenrun precision data for the DNA probe assay for matched and unmatched specimens. This interassay precision data was calculated by assaying selected samples in several consecutive runs. A negative control sample, a positive control sample, and several patient samples with various sample/cutoff ratios were evaluated. The coefficient of variation for the negative control samples was approximately 28%, whereas the coeffi-

Volume 165 Number 5, Part

DNA probe test vs culture for C. trachomatis

1447

1

'*

'*

ube/edprobe }(J(Jx lIJl1.rbe/ed CAJUlI)'di.r probe

ube/edprobe }(J(Jx llJl1.rbe/ed M)'cop/.rsm.r probe

Tube #1

Tube #2

Tube #3

Test sample Referrnce RLU

Competition >90% reduced RLU

Conlrol Reference RLU

• - Chlamydia rRNA

C\J - Single-stranded DNA probe ~

- Chemiluminescent marker Unlabeled Mycoplasma probe

w-

A

'*

Tube #1 Reference RLU

'*

ube/edproN 1(J(Jx lIJl1.r!JeJt1d CAJUl1)'dI.r probe

ube/edprobe }(J(Jx 11J1/.r!JeJed M)'cop/lISD1.r probe

Tube #2 No hybridizalion/competition <90% reduced RLU

Tube #3 Control Reference RLU

• - Chlamydia rRNA - Single-stranded DNA probe ~ - Chemiluminescent marker w - Unlabeled Mycoplasma probe

W

B

Fig. 2. Probe competition assay. A, Positive reaction occurred when >90% reduction in difference in relative light units was detected in tube No. 2 compared with tubes No. 1 and No.3. When C. trachomatis nucleic acids were present in the sample, competition reaction occurred in tube No.2. B, Negative reaction occurred when <90% reduction in difference in relative light units was detected in tube No.2 compared with tubes No.1 and No.3. No competition reaction occurred in tube No.2. cient of variation for the positive control samples was approximately 16%. The coefficients of variation for the patient samples were as high as 23%. With such interassay variation of up to 30%, occasional patient samples that were truly positive for C. trachomatis could, on a single run, have a ratio as low as 0.7. Patient samples near the cutoff value (ratio 1.0) were assayed several times to ascertain whether DNA probe retesting of those samples allowed for consistent and clinically relevant results. Samples were considered negative for

C. trachomatis by the DNA probe assay if results were consistently < 1.0 (i.e., 0.7 :S ratio < 1.0) or a single ratio 2: 1.0 (i.e., 3.0 > ratio 2: 1.0) occurred among other results < 1.0. Samples were considered positive for C. trachomatis by the DNA probe assay if results were consistently 2: 1.0 (i.e., 3.0 > ratio 2: 1.0) or a single ratio < 1.0 (i.e., 0.7 :S ratio < 1.0) occurred among other results 2: 1.0. Correlation of the DNA probe assay resnlts with those of culture and clinical data. In this study, true

1448 Yang et al.

November 1991 Am J Obstet Gynecol

Culture

Culture

+

+

DNA

Probe Test

Culture

+

18

1

L..-_3_-----'--_~

DNA

+

+

13

2

+

DNA

Probe Test

31

3

5

387

Probe Test

152

2

(a)

--

(c)

(b)

Fig. 3. Comparison of DNA probe test and tissue culture for detection of C. trachomatis. a, Pregnant patients without symptoms; b, patients with symptoms; c, combined patients with and without symptoms. True-positive samples were those specimens positive by tissue culture or by two nonculture tests (i.e., DNA probe test and probe competition assay) if tissue culture was negative.

Table I. lnterassay precision data Sample cutoff (RLU) Mean

I

SD

I

Sample

No. of DNA tests run

Coefficirnt of variation (%)

Negative control Positive control Patient identification No. 36764 35128 31731 34399 30888 35322 37775 39529 35178 38897

22* 21

0.29 4.32

0.08 0.70

27.6 16.2

5 5 6 4 6 5 4 5 5 5

0.39 0.46 0.82 1.16 2.47 5.41 6.86 9.06 15.75 33.20

0.18 0.08 0.19 0.17 0.12 0.43 0.40 0.69 1.62 2.30

19.4 17.4 23.2 14.7 4.8 8.0 5.8 7.6 10.3 6.9

*Two negative controls had values >3 SDs from the mean (negative control No. I had a ratio of 0.67; negative control No.2 had a ratio of 0.63), and they were not used to produce these results.

positive samples were considered to be those specimens that were positive by culture or positive by two noncultural tests (i.e., DNA probe test and probe competition assay) if culture was negative. The results obtained by DNA probe and culture for asymptomatic and symptomatic patients are shown in Fig 3, a and b, respectively. Fig. 3, c, depicts the data for all patients, regardless of symptoms. The number of pregnant patients without symptoms included in the study was 257 (Fig. 3, a). Twenty-two patients had positive results by culture alone or two noncultural methods, resulting in a disease prevalence of 8.6%. Four of these patients had discrepant results. One patient was DNA probe positive, but culture negative. This sample was positive for C. trachomatis after repeat DNA probe testing (i.e., ratios, 8.35, 9.32). The sample for DNA probe was also submitted in blind fashion for probe competition assay, which verified the

presence of C. trachomatis nucleic acid. The three remaining patients were all culture positive and DNA probe negative. The negative results were confirmed by DNA probe competition assay. These probe samples were also retested with the DNA probe assay and all resulted in negative ratios (i.e., patient 42315, ratios = 0.51, 0.45, and 0.28: patient 35543, ratios = 0.17 and 0.25; patient 39971, ratios = 0.48 and 0.37). DNA probe assays performed on the specimens submitted for culture were all positive. The DNA assay demonstrated 98.4% correlation with culture in asymptomatic patients. The number of symptomatic patients was 169. Seventeen patients had positive results by culture alone or two noncultural methods, with a disease prevalence of 10.1 %. Four of these patients had discrepant results (Fig. 3, b). Two patients were positive by culture and negative by DNA probe test. The samples submitted

DNA probe test vs culture for C. trachomatis

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Table II. Sensitivity, specificity, and predictive values for DNA probe and culture Sensitivity 95% Confidence interval

Specificity

(%)

Positive predictive value (%)

%

86.4 95.4

75-100 87-100

100 100

100 100

98.7 99.6

97-100 99-100

88.2 88.2

76-100 76-100

100 100

100 100

98.7 98.7

97-100 97-100

87.2 92.3

79-98 85-100

100 100

100 100

98.7 99.2

98-100 98-100

%

Group

Without symptoms DNA probe test Culture With symptoms DNA probe test Culture Combined DNA probe test Culture

Negative predictive value

I

I

95% Confidence interval

Table III. Sensitivity, specificity, and predictive values for DNA probe versus culture alone as gold standard Sensitivity DNA probe test group

%

Without symptoms Symptomatic Combined

85.7 86.7 86.1

I 95% Confidence

Specificity

interval

%

71-100 70-100 75-97

99.6 98.7 99.2

Positive predictive value

I 95% Confidence

for the DNA test were subsequently retested with the DNA probe assay. The DNA probe assay was consistently negative for both samples (i.e., patient 37640, ratios = 0.14 and 0.17; patient 28725, ratios = 0.11 and 0.38). The probe competition assays verified the absence of C. trachomatis nucleic acid in the specimens submitted. DNA probe assays performed on the specimens submitted for culture were positive. The two remaining patients were negative for C. trachomatis by culture and positive by DNA probe test. The DNA probe test was performed several times on these probe samples and consistently positive results were obtained (i.e., patient 29983, ratios = 1.31, 1.54, and 1.28; patient 26549, ratios = 3.14, 3.56, and 3.58). Probe competition assay confirmed the presence of C. trachomatis nucleic acid in the probe samples. The DNA probe assay demonstrated 97.6% correlation with culture in symptomatic patients. Fig. 3, c, shows the statistical parameters for the DNA probe test and culture when the results from the patients with and without symptoms were combined. Of the total 426 patients tested, C. trachomatis was detected in 36 (8.1%) specimens by culture and in 34 (8.0%) specimens by the DNA probe test. Overall, the DNA probe test demonstrated 98.1 % agreement with culture. For all patients combined, the disease prevalence was 10.1 %, There was no statistical difference between DNA probe test and standard cell culture results by X2 analysis. Table II depicts the sensitivity, specificity, positive

interval

%

99-100 97-100 98-100

94.7 86.7 91.2

I

Negative predictive value

95% Confidence interval

%

85-100 70-100 82-100

98.7 98.7 98.7

I

95% Confidence interval

97-100 97-100 98-100

and negative predictive values, and 95% confidence intervals for both the DNA probe test and culture in asymptomatic, symptomatic, and combined patient groups using culture or two noncultural methods as the gold standard. Table III depicts the sensitivity, specificity, positive and negative predictive values, and 95% confidence intervals for the DNA probe versus culture alone as the gold standard. Similar results were obtained for both methods of analysis. Samples that tested negative for C. trachomatis by the DNA probe assay. Table IV shows the distribution of the sample/ cutoff ratios for samples that tested negative for C. trachomatis by the DNA probe assay. With the manufacturer's recommended cutoff, 392 of 426 patients in this study were negative for chlamydia. Approximately 96% of these negative samples were at least 50% away from the cutoff value (ratio, :s0.5). Randomly selected samples from patients with ratios :s0.5 were assayed several times and remained negative (data not shown). Seventeen study patients who were negative for C. trachomatis by the DNA probe test had sample/ cutoff ratios >0.5 and < 1.0. Sixteen of these patients were negative for C. trachomatis by tissue culture, and only one patient was positive. Repeat testing of this patient by DNA probe assay was performed on the sample submitted for the DNA probe testing. This sample yielded consistently negative results for C. trachomatis (i.e., patient 27348, ratios = 0.53.0.11, and 0.24). Additionally, probe competition assay performed on this sample confirmed the lack of C. trachomatis nucleic

1450 Yang et al.

November 1991 Am J Obstet Gynecol

Table IV. Distribution of values in relative light units in patients who tested negative for C. trachomatis by DNA probe assay Study patients* (both DNA assay and culture)

%

No.

0 4 1 6 6 9 27 104 230 5

1.0 0.3 1.5 1.5 2.3 6.9 26.5 58.7 1.3

11 15 26 32 59 90 204 677 1296 38

392

100

2448

Range (sample Icutoff)

No.

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

>Ratio ~0.9 >Ratio ~0.8 >Ratio ~0.7 >Ratio ~0.6 >Ratio ~0.5 >Ratio ~0.4 >Ratio ~0.3 >Ratio ~0.2 > Ratio ~O.l > Ratio ~O.O

TOTAL

I

Additional patientst (DNA assa,V only)

I

% 0.4 0.6 1.1

1.3 2.4 3.7 8.3 27.7 52.9 1.6 100

*A total of 426 study patients were evaluated for the presence of C. trachomatis by DNA probe assay and culture. With the manufacturer's recommended cutoff, 392 (92.0%) of the study patients were negative for C. trachomatis by the DNA probe assay. t A total of 2703 patients were evaluated for the presence of C. trachomatis by DNA probe assay alone. With the manufacturer's recommended cutoff, 2448 (90.6%) of the patients were negative for C. trachomatis.

acid in the sample submitted. The DNA probe assay performed on the specimen submitted for culture was positive. An additional 2703 patient samples were analyzed by DNA probe assay alone during October 1989 and June 1990. The sample / cutoff ratios were determined (Table IV). No matching cultures were performed. Out of 2703 samples analyzed, 2448 (90.6%) specimens were negative for C. trachomatis. Approximatey 5% of patients who tested negative for C. trachomatis had sample/ cutoff ratios between 0.5 and 1.0. Eleven patients had ratios >0.9 and <1.0 after initial testing. Nine of these patients were retested by the DNA probe assay. Six patients remained negative for C. trachomatis; however, three of the nine patients were positive for C. trachomatis (i.e., patient 39891, ratios = 0.96, 1.24, and 1.72; patient 33767, ratios = 0.90,1.08, and 1.00; patient 33342, ratios = 0.98, 1.10, and 1.15). Multiple samples from patients with ratios between 0.8 and 0.9 and between 0.7 and 0.8 were assayed several times. Although all of these samples remained negative after retesting, one sample that had a ratio of 0.77 on primary testing had a single positive ratio when assayed several times (i.e., patient 29435, ratios = 0.77, 1.42, 0.99,0.67, and 0.55). Samples that tested positive for C. trachomatis by the DNA probe assay. Table V depicts sample/cutoff ratios in patient samples that tested positive for C. trachomatis by the DNA probe assay. The ratios are shown for all study patients (patients who had a DNA probe assay and a matching culture) and for 2703 patient samples evaluated only by the DNA probe assay without matching cultures. Randomly selected patients with sample/cutoff ratios 2:3.0 were retested for C. trachomatis by the DNA probe assay, and all remained positive

(data not shown). Two study patients had sample/cutoff ratios <3.0 and 2:1.0. One patient (i.e., patient 24537, ratio = 2.47) was positive for C. trachomatis by culture. The other patient tested negative by culture; however, this patient was clinically symptomatic and was positive for C. trachomatis by repeat DNA probe testing on the probe specimen (i.e., patient 25897, ratios = 1.3, 1.5, 1.2). Additionally, probe competition assay confirmed the presence of C. trachomatis nucleic acids in this patient's sample. Of the 2703 patients who had only the DNA probe test performed, 38 (1.4%) had sample/cutoff ratios < 1.5 and 2: 1.0. Twenty-six of these patients were retested for C. trachomatis by DNA probe assay. Eight patients remained positive for C. trachomatis, whereas 17 had negative ratios. In the same group of 2703 patients, 14 (0.5%) had sample/ cutoff ratios <2.0 and 2: 1.5. Eight of these patient samples were retested by the DNA probe assay. Three remained positive for C. trachomatis, whereas five had negative ratios after retesting. Finally, of the 2703 patients who had only the DNA probe test performed, 13 (0.5%) had sample/cutoffratios <3.0 and 2:2.0. Five of these patient samples were retested by the DNA probe assay, resulting in only one patient with negative ratios for C. trachomatis (i.e., patient 29987, ratios = 2.05, 0.46, and 0.98). Randomly selected samples from patients with ratios 2:3.0 were assayed several times; all samples remained positive (data not shown).

Comment Infection by C. trachomatis is limited almost exclusively to humans. 6 It is one of the most prevalent pathogens in the world and is the most common sexually

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Table V. Distribution of values in relative light units in patients who tested positive for C. trachomatis by DNA probe assay Study patients* (both DNA assay and culture) Range (sample / cutoff)

No.

Ratio ~IO.O 10.0 >Ratio 7.0 >Ratio 6.0 >Ratio 5.0 >Ratio 4.0 >Ratio 3.0 >Ratio 2.0 >Ratio 1.5 >Ratio

26 2 1 0 0 3 1 0 1

TOTAL

~7.0 ~6.0

~5.0 ~4.0

~3.0 ~2.0 ~1.5 ~1.0

I

34

Additional patientst (DNA assay only)

%

No.

76.5 5.9 2.9 0.0 0.0 8.9 2.9 0.0 2.9

137 10 9 10 8 16 13 14 38

100

255

I

% 53.7 3.9 3.5 3.9 3.1 6.3 5.1 5.5 14.9 100

*A total of 426 study patients were evaluated for the presence of C. tmchomatis by DNA probe assay and culture. With the manufacturer's recommended cutoff. 34 (8.0%) of the study patients were positive for C. tmchomatis by the DNA probe assay. t A total of 2703 patients were evaluated for the presence of C. trachomatis by DNA probe assay alone. With the manufacturer's recommended cutoff, 255 (9.4%) of the patients were positive for C. trachoma/is.

transmitted disease in developed countries. Cell culture has remained the standard for diagnosis, although some studies have estimated that a single endocervical specimen may be only 70% to 90% sensitive for C. trachomatis infection." Ii Cell culture is technically difficult and requires organism viability and timely transport of specimens to the laboratory for processing. In addition, cell culture is labor intensive, requires trained personnel, and has a routine turnaround time of 48 hours. Demand for simplified and rapid methods of diagnosis has led to development of many antigen detection methods for C. trachoma tis infection over the past 10 years. These methods are based on immunodetection of solubilized chlamydia components (enzyme immunoassay) or direct visualization of organisms with chlamydia-specific fluorescein-conjugated monoclonal antibodies (direct fluorescent antibody). Studies comparing these noncultural methods to standard cell culture report sensitivities of enzyme immunoassay of 67% to 98% and sensitivities for direct fluorescent antibody of 72% to 99%.7.10 Barnes" recently reviewed several studies from 1984 to 1988 comparing enzyme immunoassay with cell culture. These studies demonstrated a range of sensitivity from 70% to 100% and a positive predictive value from 6% to 92%. The prevalence of endocervical infection ranged from 5% to 40%. Similar comparison of direct fluorescent antibody to cell culture in populations with a prevalence of 5% to 27% showed sensitivities from 56% to 100% and positive predictive values from 47% to 100%. Because of the wide range of sensitivities reported in many studies, antigen detection tests have met with mixed acceptance by clinicians for the detection of chlamydial infection. Recently, attention has turned to DNA probes for the detection of infection, and favorable results have been reported with sensitivities of 92%.11

Our study population had a prevalence of infection of 8.6% and 10.1 % for asymptomatic and symptomatic patients, respectively. The prevalence of infection in asymptomatic women is comparable to previous reports of 7% to 11 %.7.11. 12 The infection rate in the symptomatic group is lower than expected but comparable to previous reports of 14% to 20%.9. II Our symptomatic group probably does not represent a truly high-risk subpopulation. The DNA probe test performed well in our relatively low-prevalence population. Comparison of nonculture tests, such as the DNA probe, with culture methods of < 100% sensitivity must be interpreted with caution. If culture alone is used as the gold standard, then culture would necessarily have 100% sensitivity. Positive DNA probes might be misinterpreted as being falsely positive rather than culture results being falsely negative. We attempted to address this issue by sending discrepant specimens to another laboratory for DNA probe competition assays. Probe competition assays are reported by Cen-Probe as having 100% correlation with other hybridization techniques. Our results were blinded to the outside laboratory. In this study we defined the number of true positives to be the sum of specimens positive by culture or positive by a DNA probe assay, with confirmation of the presence of C. trach01natis nucleic acid in the sample by probe competition assay. Although this is not standard analysis, other authors have attempted to address this issue similarly with noncuItural method confirmation. 7. 9 We did include standard analytic parameters of test validity with culture alone used as the gold standard (Table III), and, comparable results for the DNA probe were obtained. Overall, the DNA probe performed well with 98% agreement with culture in our study. In the asymptomatic population, three patients were

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DNA probe negative but culture positIve for C. trachomatis. DNA probe competition assay was negative in all these samples, indicating that rRNA for C. tmchomatis was not present in the specimens submitted for the DNA probe assay. DNA probe tests done on the culturepositive specimens were also positive, indicating the presence of probe-detectable DNA sequences. The one patient who was positive for C. trachomatis by DNA probe test and negative by culture was found to have C. trachomatis nucleic acid present in the probe sample by probe competition assay. The DNA probe assay gave no false-positive results in this patient population. We obtained similar results with our patient population with symptoms. Two patients were DNA probe negative and culture positive. DNA probe competition assays did not indicate the presence of C. trachomatis rRNA. Again, DNA probe tests done on culture-positive specimens were positive indicating the presence of probe-detectable DNA sequences. Two patients were DNA probe positive and culture negative. Probe competition assays verified the presence of C. trachomatis; therefore these samples were considered true positives. Overall, the tissue culture method produced three false-negative results, whereas DNA probe assay produced five false negatives. Tissue culture technique can have a large impact on the ability to isolate C. trachomatis. Single passage with shell vials and immunofluorescence staining is standard technique and has been shown to yield high sensitivities. 13 Automatic passage of tissue culture specimens has been reported to increase the sensitivity of this method by approximately 3% to 10%.6,14 However, passage with this technique is not commonly practiced in many large clinical institutions. Multiple passages in shell vial systems, in contradistinction to microtiter plates. has not been shown to increase sensitivity of culture. 14 For these reasons, automatic passage was not done on tissue culture specimens in this study. All discrepant or suspicious specimens underwent a second passage with confirmation of initial results. Sampling error is the most likely source of discrepant results for both culture and DNA probe assay. Patient specimens for culture and for the DNA probe test were collected with separate swabs. In previous culture versus nonculture evaluations, discrepancy rates of 5% to 13% have been reported as a result of sample variation between multiple swabs. 15 Detection of chlamydia by any method requires the sampling of endocervical cells. Obtaining several swabs and removal of excess mucus has been observed to increase detection rate. 15, Hi One problem we found with the DNA probe test was the discrete manufacturer recommended cutoff of 300 RLU above the mean of the negative references. The results of 426 culture-matched samples suggested that the manufacturers' recommended discrete cutoff was

November 1991 Am J Obstet Gynecol

adequate for determination of results. Our results on 2703 patient samples without matching cultures indicated that samples with sample/cutoff ratios of 0.7 to 3.0 should have repeat testing until unequivocal results are obtained. Out of the 3129 patient samples evaluated by the DNA probe assay in this paper. 124 patients (4.0%) fell into this range. Further study with matching cultures would be helpful to confirm this finding. However, without modification the DNA probe test was equivalent to standard tissue culture in sensitivity. specificity, and positive and negative predictive values for our 426-patient study population, when culture alone or two noncultural methods were used as the gold standard. The fact sheet from the National Institute of Allergy and Infectious Disease in 1986 recommended that all sexually active women <35 years old be tested yearly for C. trachomatis. 17 The DNA probe is a rapid, costeffective method of screening women for infection with 87.2% sensitivity in our low-risk populations. Infection by chlamydia can potentially result in serious long-term sequelae, including ectopic pregnancy, infertility, chronic pelvic pain, and pelvic inflammatory disease. Vertical transmission from mother to fetus has been well established. Recent studies implicate chlamydial infection with increased risk of premature rupture of the membranes, low birth weight, and preterm delivery.' 4,18 Rapid tests for diagnosis of chlamydial infection in obstetrics would be of benefit to mother and fetus. Treatment is simple and effective once the diagnosis is made with laboratory confirmation. Positive test results improve diagnostic correlation for the physician and patient treatment compliance and enhance the likelihood that her sex partner will seek referral for evaluation. Coinfecton rate with Neisseria gonorrhoeae can be as high as 50%.1 The Centers for Disease Control currently recommend presumptive treatment for both organisms if one is detected. '0 Specific diagnosis can direct antibiotic therapy for patients and their partners. The DNA probe test also can be used for the detection of both organisms from the same endocervical sample. and results can be made available in approximately 2 hours. In addition, the DNA probe can be a costeffective alternative to cell culture. Our study indicates that the PACE 2 DNA probe is a reasonable alternative to standard cell culture for the rapid diagnosis of chlamydial infection in women. Future DNA probe technology likely will be applied to many other types of infection. The technical assistance of Leslie Leopold, Mary Rath. Rosemary Gantzer, and the Microbiology Medical Technology Staff is deeply appreciated. We also acknowledge Beth Lucas and Patricia Magevney for coordinating the acquisition of patient samples and their clinical histories. We thank Rick Helmchen and

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Kimberly Hasselfeld for assistance in data analysis and critical review of the manuscript.

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REFERENCES I. Centers for Disease Control. Chlamvdia trachomatis infections: policy and control. MMWR (985;34(suppl. 3):53s74s. 2. Sweet RL, Schachter j, Landers D. Chlamydial infections in obstetrics and gynecology. Clin Obstet Gynecol 1983;26: 143-64. 3. Sweet RL, Landers D, Walker C, Schachter j. Chlam,vdia trachomatis infection and pregnancy outcome. AM j OBSTET GYNECOL 1987;156:824-33. 4. Ryan GM, Abdella TN, McNeeley GS, Baselski VS, Drummond DE. Chlamydia trachomatis infection in pregnancy and effect of treatment outcome. AM j OBSTET GYNECOL 1990;162:34-9. 5. Arnold Lj, Hammond PW, Wiese WA, Nelson NC. Assay formats involving acridinum-ester-Iabeled DNA probes. Clin Chern 1989;35: 1588-94. 6. Barnes RC. Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 1989;2:119-36. 7. Lefebevre j, Laperriere H, Rousseau H, Masse R. Comparison of three techniques for detection of Chlamydia trachomatis in endocervical specimens for asymptomatic women. j Clin Microbiol 1988;26:726-31. 8. LeBar W, Herschman B, jemal C, Pierzchala J. Comparison of DNA probe, monoclonal antibody enzyme immunoassay and cell culture for the detection of Chlamydia trachomatis. j Clin Microbiol 1989;27:826-8. 9. Hipp SS, Han Y, Murphy D. Assessment of enzyme immunoassay and immunofluorescence tests for detection

II.

12. 13.

14. 15.

16. 17. 18. 19.

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of Chlamydia trachomatis. j Clin Microbiol 1987;25:193843. Smith jW, Rogers RE, Katz BP, et al. Diagnosis of chlamydial infection in women attending antenatal and gynecologic clinics. j Clin Microbiol 1987;25:868-72. Pao CC, Lin S, Yang T, Soong Y, Lee P, Lin J. Deoxyribonucleic acid hybridization analysis for the detection of urogenital Chlamydia trachomatis infections in women. AM j OBSTET GYNECOL 1987;156:195-9. Stamm WE. Diagnosis of Chlamydia trachomatis genitourinary infections. Ann Intern Med 1988; 108:710-7. Neinstein LS, Rabinovitz S. Detection of Chlamydia trachomatis: a study of the direct immunofluorescence technique and a review of diagnostic limitations. j Adolesc Health Care 1988;10:10-5. Schachter j, Martin DH. Failure of multiple passages to increase chlamydial recovery. j Clin Microbiol 1987; 25: 1851-3. Embil .lA, Thiebaux H.l, Manuel FR, Pereira LH, McDonald SW. Sequential cervical specimens and the isolation of Chlamydia trachomatis: factors affecting detection. Sex Transm Dis 1982; 10:62-6. Munday PE, Carder .lM, Hanna NF, Taylor-Robinson D. Is one swab enough to detect chlamydial infection of the cervix? Br j Vener Dis 1984;60:385-6. Medical News and Perspectives. Sexually transmitted diseases may reverse the revolution. .lAMA 1986;255:16672. Heggie AD, Lumicao GG, Stuart LA, Gyres MT. Chlamydia trachomatis infection in mothers and infants. Am .l Dis Child 1981;135:507-11. Centers for Disease Control. Sexually transmitted diseases treatment guidelines. MMWR 1989;38(suppl 8): 15s-43s.

Editors' note The AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY introduces a new format for abstracts accompanying regular articles, society articles, and Current Investigation articles. Authors submitting these manuscripts to the JOURNAL should provide an abstract of no more than 150 words structured according to the following headings: Objective(s), Study Design, Results, and Conclusion(s). Exceptions to this requirement include Clinical Opinion, Current Development, case report, and brief communication articles. Abstracts for these articles will continue to follow the standard abstract format. Please consult the Information for Authors for details.