Comparison of Manual Amplicor PCR, Cobas Amplicor PCR, and LCx ...

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1997, p. 402–405 0095-1137/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 35, No. 2

Comparison of Manual Amplicor PCR, Cobas Amplicor PCR, and LCx Assays for Detection of Chlamydia trachomatis Infection in Women by Using Urine Specimens ¨ JA ¨ RVI,2 MARKKU KOSKELA,3 RAFAEL PASTERNACK,1* PAULI VUORINEN,1 TIMO PITKA 1 AND ARI MIETTINEN Department of Clinical Microbiology, Tampere University Hospital, FIN-33521 Tampere,1 Tampere Community Health Centre, FIN-33100 Tampere,2 and Department of Clinical Microbiology, Oulu University Hospital, FIN-90221 Oulu,3 Finland Received 1 July 1996/Returned for modification 13 August 1996/Accepted 31 October 1996

We compared the Roche Amplicor PCR, Roche Cobas Amplicor PCR, and Abbott LCx assays by using urine specimens for the detection of Chlamydia trachomatis infections in a female population. First-catch urine and endocervical swab specimens were collected from a total of 442 patients. Urine specimens were tested by the manual Roche Amplicor PCR, the automatic Roche Cobas Amplicor PCR, and the Abbott LCx assays as instructed by the manufacturers. For the Cobas Amplicor PCR, the internal control protocol was used for every specimen to reveal the presence of polymerase inhibitors. Cell culture of cervical specimens was used as a reference method. Of 442 patients, 50 (11.3%) were confirmed to have chlamydial infection. The diagnostic sensitivity and specificity of cell culture with cervical swab specimens were 88 and 100%, respectively. With urine specimens the sensitivity and specificity for the manual Amplicor PCR assay were 100 and 99.7%, respectively; those for the automatic Cobas Amplicor PCR assay were 94 and 99.2%, respectively; and those for the LCx assay were 94 and 100%, respectively. Thus, all amplification methods with urine specimens proved to be highly sensitive and specific for the detection of C. trachomatis infection in women. No statistically significant differences in the test performances could be demonstrated for specimens from this population. All three amplification techniques with urine specimens proved to be superior to cell culture with cervical swab specimens in diagnosing C. trachomatis infections in women. The introduction of assays based on amplification of genetic material has substantially increased the sensitivity of chlamydial detection. The detection of Chlamydia trachomatis from first-catch urine by PCR and ligase chain reaction (LCR) has been demonstrated to be at least as sensitive as cell culture for both male and female populations (1, 3, 4, 7). Two automatic applications of amplification assays for the detection of C. trachomatis, the Roche Cobas Amplicor PCR and Abbott LCx assays, have recently been introduced. They are expected to standardize the performance of nucleic acid amplification tests in routine clinical microbiology laboratories. So far, no direct comparisons between the automatic amplification methods have been published. We evaluated these assays by using female urine specimens and compared them with the manual Roche Amplicor PCR assay by using urine specimens and cell culture with cervical specimens. To evaluate the frequency of DNA polymerase inhibition in urine specimens, the internal control protocol was used in conjunction with the Cobas Amplicor PCR assay.

specimens were transported to the laboratory within 24 h. Once at the laboratory, urine specimens for the Amplicor PCR, Cobas Amplicor PCR, and LCx assays were prepared as instructed by the manufacturers. The Roche Amplicor PCR assay (F. Hoffmann-La Roche Ltd., Basel, Switzerland) with urine specimens was performed according to the manufacturer’s instructions. PCR was accomplished with the Perkin-Elmer Thermocycler TC 9600 instrument (Perkin-Elmer Cetus, Norwalk, Conn.). After the amplification, the amplified nucleotide sequences were detected by using target-specific DNA probes. The resulting enzyme reaction was measured with a spectrophotometer (Multiskan; Labsystems Ltd., Helsinki, Finland). Specimens with an A450 of $0.250 were considered positive. The Cobas Amplicor CT/NG PCR assay (F. Hoffmann-La Roche Ltd.) with urine specimens was performed according to the manufacturer’s instructions. In brief, the prepared specimens were transferred into amplification tubes containing primers for C. trachomatis and Neisseria gonorrhoeae, nucleotides, the internal control, and DNA polymerase. The amplification was accomplished by the built-in thermocycler. The internal control of amplification in the Cobas Amplicor CT/NG assay is a sequence of plasmid DNA with primer binding regions identical to those of the C. trachomatis target sequence and a randomized internal sequence with a length and base composition similar to those of the C. trachomatis target sequence. A unique probe binding region differentiates the internal control from the target amplicon. The internal control is introduced into each amplification reaction and is coamplified with the possible target DNA from the clinical specimen. After the amplification, the amplified nucleotide sequences for the internal control and C. trachomatis were automatically detected by using target-specific DNA probes, and the A660s were measured by the built-in spectrophotometer. N. gonorrhoeae sequences were not detected. The urine LCR assay, the LCx Chlamydia trachomatis (Abbott Laboratories, Chicago, Ill.) assay, was performed according to the instructions of the manufacturer and described earlier (4). In brief, 100 ml of the processed urine specimen was transferred to the unit-dose tubes. The amplification was accomplished by the LCx system thermocycler. After the amplification, the tubes containing the amplified nucleotide sequences were pulse centrifuged and manually transferred to LCx reaction cells. The detection was performed by the LCx analyzer. Endocervical swab specimens were obtained by using TS/19-CW aluminum wire swabs with a viscose tip (Technical Service Consultants Ltd., Heywood, United Kingdom). After removal of the mucus from the cervix, the swab was revolved in the endocervix to collect epithelial cells. The swab was then shaken vigorously with the glass beads in chlamydia transit tubes (Labsystems Ltd.) and compressed toward the tube wall to free the specimen from the swab. The specimens were transported to the laboratory within 24 h. The cell culture was

MATERIALS AND METHODS The patients consisted of 442 women. They were examined and treated at the Policlinic for Sexually Transmitted Diseases, Community Health Centre of the City of Tampere, Finland, and at the policlinic of the University Student’s Health Foundation, Tampere, Finland, during the period from October 1995 to April 1996. The study protocol had been approved by the ethical committees of Tampere University Hospital and the City of Tampere. Informed consent was obtained from all patients verbally. First-catch urine specimens (20 to 50 ml) were collected in a clean cup prior to the cervical sampling and were transferred to a sterile plastic container. The

* Corresponding author. Mailing address: Department of Clinical Microbiology, Tampere University Hospital, Box 2000, FIN-33521 Tampere, Finland. Phone: 358-3-2475296. Fax: 358-3-2475260. 402

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TABLE 1. Analysis of discrepant test results by Roche Amplicor PCR, Roche Cobas Amplicor PCR, and Abbott LCx assays and chlamydial cell culture Result by: No. of specimens

Amplicor PCR with urine specimens

Cobas Amplicor PCR with urine specimens

LCx with urine specimens

Cell culture with cervical swab specimens

Final result and conclusion

6

Positive

Positive

Positive

Negative

3

Positive

Negative

Positive

Positive

3

Positive

Positive

Negative

Positive

3

Negative

Positive

Negative

Negative

1

Positive

Negative

Negative

Negative

Positive; the infection was not detected in the cervix by cell culture or Amplicor PCR Positive; the infection was not detected in urine by Cobas Amplicor PCR Positive; the infection was not detected in urine by LCx assay Negative; the positive result could not be repeated or confirmed by MOMP PCR Negative; the repeatedly positive result by Amplicor PCR could not be confirmed by MOMP PCR; the patient was symptomatic and was treated with doxycycline based on clinical decision

performed by inoculation of 500 ml of the specimen in sucrose-phosphate medium onto a monolayer of McCoy cells in flat-bottom, plastic shell vials with coverslips. The specimens were centrifuged (3,000 3 g) at 358C for 1 h. The medium was then changed to a medium containing 1 mg of cycloheximide per ml (9). After incubation for 3 days, the cells were stained with iodine and were screened for inclusions. If one or more typical inclusions were seen, the specimen was interpreted as positive. Previous studies in this laboratory (7) indicate sensitivity for this procedure to be 85%. Cell culture-negative endocervical specimens from patients whose urine was positive by any of the amplification tests were also tested by the Roche Amplicor PCR, which was performed according to the manufacturer’s instructions. Urine specimens that were positive by PCR only were subjected to a confirmatory PCR assay with major outer membrane protein (MOMP)-based primers as described previously (2). The MOMP PCR was performed by Roche Laboratories (Roche Molecular Systems, Basel, Switzerland), which was provided with a panel of discrepant specimens including known positive and known negative specimens, and the MOMP PCR was performed by investigators who were blinded to the original results. A positive culture of a cervical swab specimen was taken to indicate C. trachomatis infection. In addition, patients whose endocervical swab specimens were negative by cell culture were determined to have C. trachomatis infection if their urine tested positive by PCR and LCR with urine or if their urine was positive by one of the two PCR applications and the MOMP PCR. Reliability was measured by using the kappa coefficient, which compares the agreement of these tests in detecting the total number of infections. Values greater than 0.75 were taken to indicate strong agreement between the tests.

BMDP (version 7.01; BMDP Statistical Software, Inc., Los Angeles, Calif.) was used for the statistical analysis.

RESULTS The results of all four tests were identical for 426 (96.4%) of the 442 patients. Altogether 50 (11.3%) of the patients had chlamydial infection by definition. Among them, the test results were uniformly positive for 38 patients. The discrepant results for 16 patients are presented in Table 1. Of them, 12 could be confirmed to be positive. The diagnostic sensitivity and specificity as well as the positive and negative predictive values of the different assays are presented in Table 2. The kappa coefficient value for cell culture was 0.929, that for the Amplicor PCR was 0.989, that for the Cobas Amplicor PCR was 0.932, and that for the LCx assay was 0.965, indicating strong agreement between the tests. The internal control coamplified by the Cobas Amplicor PCR revealed PCR inhibitors in 13 (3%) of the 442 urine specimens. When tests with these specimens were rerun with the specimens undiluted and diluted 1:10, they remained neg-

TABLE 2. Comparison of Roche Amplicor PCR, Roche Cobas Amplicor PCR, and Abbott LCx assays with urine specimens and cell culture with cervical swab specimens for the detection of genital C. trachomatis infections in 442 women Test (specimen) and result

No. of patients in whom C. trachomatis infection was:

Sensitivity (%)

Specificity (%)

Positive predictive value (%)

99.7

98.0

99.2

92.2

Negative predictive value (%)

Present (n 5 50)

Absent (n 5 392)

Amplicor PCR (urine) Positive Negative

50 0

1 391

Cobas Amplicor PCR (urine) Positive Negative

47 3

3 389

94.0

LCx Chlamydia (urine) Positive Negative

47 3

0 392

94.0

100

100

99.2

Cell culture (cervix) Positive Negative

44 6

0 392

88.0

100

100

98.5

100

100

99.2

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PASTERNACK ET AL.

ative and were determined to be true negatives. They were also negative by the LCx assay. Three urine specimens from patients who were positive by all other methods were negative by the LCx assay with urine. When the prepared urine specimens were rerun in an analysis of specimens with discrepant results, two of the three specimens turned positive, indicating the presence of LCR inhibitors. When the original deep-frozen urine specimens from these patients were reanalyzed after sample preparation, the presence of LCR inhibitors was reestablished, rendering the specimens repeatedly false negative by the LCx assay. DISCUSSION The results of this study support earlier findings suggesting that urine is a superior sample material for the detection of C. trachomatis in women (4, 7, 10). In the present patient material, all 50 chlamydial infections could be detected by one or more of the amplification assays with urine specimens. In contrast, 6 (12%) of the 50 infections were not found by cell culture with endocervical specimens. In the analysis of samples with discrepant results, the six culture-negative cervical swab specimens were confirmed to be negative by the Amplicor PCR performed with the same specimens, indicating that the organism apparently was not detectable in the cervix by any method. In fact, the proportion of C. trachomatis infections that were not detectable by cervical sampling in this study is very close to that of 15 to 25% suggested in the early reports by Paavonen and Vesterinen (6) and Mårdh et al. (5), who evaluated the performance of cell culture with urethral and cervical swab specimens. It is evident from the current findings that by using any of the three nucleic acid amplification methods, the cervical samples from women can be replaced with a single urine specimen. The sensitivity thus obtained is at least the same as that by cell culture with a cervical swab specimen. Although no statistically significant differences in sensitivity and specificity were shown between the three amplification tests, the manual Amplicor PCR with urine specimens was the only test that was able to detect all positive patients. Thus, it managed better than either of the two automated methods. In addition, one patient who was repeatedly positive by the Amplicor PCR could not be confirmed to be positive by definition, although her clinical symptoms were taken to indicate chlamydial infection and she was treated with doxycycline on the basis of a clinical judgment. The Cobas Amplicor PCR missed three positive specimens. The lower sensitivity of the Cobas Amplicor PCR compared with that of the manual Amplicor PCR might be due to the smaller volume of urine or the different sample preparation protocol used in the automated assay. It is also possible that the coamplification of N. gonorrhoeae and the internal control with C. trachomatis by the Cobas Amplicor assay compromised its sensitivity. The fact that three urine specimens were false positive by the Cobas Amplicor PCR and were confirmed to be negative in the analysis of specimens with discrepant results also indicates a need for further evaluation and technical improvement of this recently introduced test system. The sensitivity of the LCx assay was also slightly lower than that of the manual Amplicor PCR. The three positive urine specimens that were missed by the LCx assay were different from those missed by the Cobas Amplicor PCR, and only one of them was missed due to suboptimal sensitivity. In two urine specimens that were false negative by the LCx assay the presence of LCR inhibitors was demonstrated. They represented 4% of the positive urine specimens, which was almost equal to the 3% frequency of PCR inhibitors in all urine specimens

J. CLIN. MICROBIOL.

tested, as detected by the internal control in the Cobas Amplicor PCR. In our earlier study, the frequency of false-negative results due to PCR inhibitors in female urine specimens was as high as 15% (7). Similarly, in other studies in which LCR was used, a strikingly higher frequency of false-negative results has been reported, which can most likely be accounted for by the presence of LCR inhibitors in 7.3 to 19% of positive urine specimens (8, 11). Theoretically, the LCR which uses both DNA ligase and DNA polymerase in the amplification reaction should not be less vulnerable than the PCR to inhibitory substances. Our results indicate that the frequency of occurrence of PCR and/or LCR inhibitors varies among different populations and even among individuals in the same geographical population at different times. Considering our earlier experiences with comparable patient populations, the frequency of occurrence of inhibitory substances in the urine specimens tested in this study was unexpectedly low. The patient material thus provided few difficulties for the amplification tests and made it hard to establish the actual value of the internal control procedure that was accomplished in conjunction with the Cobas Amplicor PCR. Nevertheless, in routine clinical microbiology laboratories the monitoring of PCR inhibitors by constant use of an internal control seems highly advisable. In conclusion, all three amplification methods with urine specimens proved to be highly sensitive and specific and superior to cell culture with cervical swab specimens for the detection of C. trachomatis infection in women. Both automated amplification tests, the Cobas Amplicor PCR and LCx assays, can be recommended for use in routine clinical microbiology laboratories because they are likely to make the testing based on nucleic acid amplification more reliable and less laborious. However, improvements in their performance are still to be expected. Further evaluation is needed to establish the necessity of using an internal control in conjunction with different amplification tests with specimens from different patient populations. ACKNOWLEDGMENT The study was supported by a grant from the Medical Research Fund of Tampere University Hospital, Tampere, Finland. REFERENCES 1. Bauwens, J. E., A. M. Clark, M. J. Loeffelholz, S. A. Herman, and W. E. Stamm. 1993. Diagnosis of Chlamydia trachomatis urethritis in men by polymerase chain reaction assay of first-catch urine. J. Clin. Microbiol. 31:3013– 3016. 2. Dutilh, B., C. Bebear, P. Rodriguez, A. Verkis, J. Bonnet, and M. Garret. 1989. Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using a polymerase chain reaction. Res. Microbiol. 140: 7–16. 3. Jaschek, G., C. A. Gaydos, L. E. Welsh, and T. C. Quinn. 1993. Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. J. Clin. Microbiol. 31:1209–1212. 4. Lee, H. H., M. A. Chernesky, J. Schachter, J. D. Burczak, W. W. Andrews, S. Muldoon, G. Leckie, and W. E. Stamm. 1995. Diagnosis of Chlamydia trachomatis genitourinary infection in women by ligase chain reaction assay of urine. Lancet 345:213–216. 5. Mårdh, P. A., L. Westro ¨m, S. Colleen, and P. Wølner-Hanssen. 1981. Sampling, specimen handling, and isolation techniques in the diagnosis of chlamydial and other genital infections. Sex. Transm. Dis. 8:280–285. 6. Paavonen, J., and E. Vesterinen. 1982. Chlamydia trachomatis in cervicitis and urethritis in women. Scand. J. Infect. Dis. 32:45–54. 7. Pasternack, R., P. Vuorinen, A. Kuukankorpi, T. Pitka ¨ja ¨rvi, and A. Miettinen. 1996. Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens. J. Clin. Microbiol. 34:995–998. 8. Ridgway, G. L., G. Mumtaz, A. J. Robinson, M. Franchini, C. Carder,

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