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Journal of Clinical Microbiology, June 2000, p. 2416-2418, Vol. 38, No. 6
Department of Pathology, Division of
Laboratory Medicine, Washington University School of
Medicine,1 and Barnes-Jewish
Hospital,2 St. Louis, Missouri 63110
Received 7 December 1999/Returned for modification 3 March
2000/Accepted 13 March 2000
This study demonstrates that significant reproducibility problems
can occur during routine use of the Abbott Laboratories LCx assay for
Chlamydia trachomatis and Neisseria
gonorrhoeae. These problems can go undetected by the quality
control procedures outlined in the manufacturer's package insert. We
outline here procedures for detecting and preventing contamination and
reproducibility problems.
The LCx assay for Chlamydia
trachomatis and Neisseria gonorrhoeae (Abbott
Laboratories, Abbott Park, Ill.) uses the ligase chain reaction
amplification method for the detection of microbial DNA. This method
has been demonstrated in numerous reports to have increased sensitivity
compared to culture (2, 4, 8, 9). In this study, the
reproducibility of the LCx assay was evaluated according to
manufacturer's recommendations in a microbiology laboratory.
The Barnes-Jewish Hospital (BJH) microbiology laboratory performs
~1,700 C. trachomatis and 1,700 N. gonorrhoeae
tests each month. Samples are obtained from a variety of geographical
locations, including the BJH emergency department, adult and adolescent
obstetrics and gynecology clinics, private physicians' offices, and a
juvenile detention hall. The majority of specimens are cervical or
urethral swabs, with 8% urine specimens. All procedures were performed as outlined in the LCx manufacturer's package insert. In addition to
the two Abbott negative control and two calibrator samples provided in
the assay kit, positive controls derived from strains of C. trachomatis and N. gonorrhoeae were also prepared and
processed as urine samples during each assay.
Abbott recommends that if a test result is in the equivocal range (0.8 to 0.99 sample/cutoff [S/CO] ratio for C. trachomatis and
0.8 to 1.2 S/CO ratio for N. gonorrhoeae) the LCx test
should be repeated. If the repeat test S/CO ratio is During the study period, 14,420 tests were performed (7,214 C. trachomatis tests and 7,206 N. gonorrhoeae tests). Of
these, 181 specimens were tested again. Of the 181, 29 (16%) changed from an initial positive result to a negative result. One additional urine specimen initially tested negative for C. trachomatis
but after a culture sample was found to be positive and was called to
our attention by the physician, the LCx testing was repeated, and the
result was positive. Thereafter, all urine samples were run in
duplicate. The data in Fig. 1 illustrate
that the magnitude of the reproducibility problem was greater in urine
samples compared to swab samples. The mean difference between the first
and second result was a 2.0 S/CO ratio for urine samples and a 0.88 S/CO ratio for swab samples. Only 5 (16%) of the 30 discrepant
specimens would have been identified by Abbott's recommended equivocal
ranges.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Reproducibility Problems with the Abbott
Laboratories LCx Assay for Chlamydia trachomatis and
Neisseria gonorrhoeae
and
ABSTRACT
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1.0, the result is considered positive, and if it is <1.0, it is reported as negative. For this study, we examined reproducibility within an expanded equivocal range (0.8 to 2.0 S/CO ratio for C. trachomatis
and 0.8 to 3.0 S/CO ratio for N. gonorrhoeae) for a 5-month
period. All repeat testing was performed within a 1-week period. Based on preliminary data, approximately 3.5 months after these changes were
instituted, the procedure was modified so that all urine samples were
tested in duplicate.
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FIG. 1.
Difference between initial and repeat testing for
C. trachomatis and N. gonorrhoeae using the
Abbott LCx. The dotted line represents the cutoff for positive and
negative results. The area between the solid lines represents the
Abbott Laboratories recommended equivocal range.
Repeated testing of the 29 specimens with initially elevated results suggested that these samples were in fact negative and that contamination or procedural error(s) likely caused the initial result. This suspicion was corroborated when known negative samples, such as urine diluent, were processed as normal urine specimens and occasionally (13%) resulted in S/CO values of >1.00. It should be noted that, during this period, the Abbott negative control and calibrator samples were performing within acceptable limits. Furthermore, monthly monitoring of work surfaces was performed by wipe testing, as outlined in the LCx package insert. Environmental testing was performed after routine cleaning with 20% (vol/vol) sodium hypochlorite solution followed by 70% ethanol, and all counts were negative.
After repeated discussions with Abbott Laboratories and other
laboratories performing the LCx assay, it became clear that these types
of problems are not uncommon. We concluded that our reproducibility
problems originated primarily in the sample processing area. Based on
suggestions from Abbott Laboratories and other LCx users, the BJH
microbiology laboratory underwent numerous changes in its specimen
processing and has incorporated procedures that are not listed in the
LCx package insert. The important changes are summarized in Table
1.
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Since implementing these changes, the BJH laboratory has had a
significant improvement in reproducibility (Table
2). In a 2-month period following
implementation of the procedure changes, 7,728 LCx tests were performed
(3,891 C. trachomatis and 3,837 N. gonorrhoeae
tests). Of these, 39 swab samples were repeated, and 475 urine
specimens were performed in duplicate (514 total). The initial result
for 13 (2.5%) of the 514 specimens differed from the second result
(C. trachomatis, 10 swabs and 1 urine; N. gonorrhoeae, 1 swab and 1 urine). Only 6 of the 13 (46%)
discrepant specimens would have been identified by Abbott's
recommended equivocal range, which supports the use of an expanded
equivocal range. The mean difference between the first and second
result was a 0.49 S/CO ratio for swab samples, which was significantly
lower (P < 0.05) than before the changes, indicating
an increase in precision. The frequency of the reproducibility problems
in urine samples was greatly reduced compared to before the processing changes. Although a rare occurrence (2 in 475), processing urine specimens remains a problem. Repeated testing of the C. trachomatis urine specimen with widely discrepant values indicated
that this specimen was clearly positive. The low value was likely due
to the presence of residual urine inhibitors (1, 3, 5) or aspiration of the centrifuged pellet. Urine specimens should be closely
monitored, including repeat testing of all samples if this is a
low-volume specimen.
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The BJH laboratory has adopted a policy that a test yielding any pair of results that do not agree should be repeated a third time, and the results should be discussed with the physician. It should be noted that these samples are not routinely confirmed by an alternative amplification method. Therefore, it is impossible to ascertain the true positive or negative status of the specimens in this study.
The addition of these quality control procedures has serious efficiency and cost implications that must be taken into consideration when selecting this test procedure. The Abbott LCx carousel is capable of holding 24 reaction cells. It is promoted as being able to perform 20 tests per run, with two spaces for negative controls and two spaces for calibrators. The instrument requires that these four controls be performed, and it cannot calculate results without them. Additionally, Abbott suggests that a positive control sample be used, although this is not provided. Furthermore, we have found that the use of two negative control specimens, processed like patient specimens, has helped us detect low levels of contamination. Therefore, a total of seven controls and only 17 patient samples are tested on each run. This decreases efficiency and increases the cost of each patient result.
The Abbott LCx provides a highly sensitive method for the detection of C. trachomatis and N. gonorrhoeae. However, as with all amplification methods, the performance of the assay must be monitored carefully for contamination and reproducibility problems. These problems are not unique to the ligase chain reaction and have been reported for other amplification tests (6, 7). Until more automated procedures evolve, problems such as we have reported here should be anticipated for all amplification assays.
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ACKNOWLEDGMENTS |
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We thank all the BJH technologists and, in particular, Sharon Nauman for their hard work and patience during the period of this study. In addition we thank Pat Plier from Abbott Laboratories.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110. Phone: (314) 362-0194. Fax: (314) 362-1461. E-mail: gronowski{at}pathology.wustl.edu.
Present address: Department of Medical Informatics, Columbia
University Medical Center, New York, N.Y.
Present address: Department of Pathology, University of Maryland,
Baltimore, Md.
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REFERENCES |
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Abstract
Text
References
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| 1. | Berg, E. S., G. Anestad, H. Moi, G. Storvold, and K. Skaug. 1997. False-negative results of a ligase chain reaction assay to detect Chlamydia trachomatis due to inhibitors in urine. Eur. J. Microbiol. Infect. Dis. 16:727-731. |
| 2. | Buimer, M., G. J. J. Doornum, S. Ching, P. G. H. Peerbooms, P. K. Plier, D. Ram, and H. H. Lee. 1996. Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by ligase chain reaction-based assays with clinical specimens from various sites: implications for diagnostic testing and screening. J. Clin. Microbiol. 34:2395-2400[Abstract]. |
| 3. | Chernesky, M. A., D. Jang, J. Sellors, K. Luinstra, S. Chong, S. Castriciano, and J. B. Mahony. 1996. Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis-infected women. Mol. Cell. Probes 11:243-249. |
| 4. |
Kehl, S. C.,
K. Georgakas,
G. R. Swain,
G. Sedmak,
S. Gradus,
A. Singh, and S. Foldy.
1998.
Evaluation of the Abbott LCx assay for detection of Neisseria gonorrhoeae in endocervical swab specimens from females.
J. Clin. Microbiol.
36:3549-3551 |
| 5. |
Mahony, J.,
S. Chong,
D. Jang,
K. Luinstra,
M. Faught,
D. Dalby,
J. Sellors, and M. 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 |
| 6. |
Mulcahy, G. M.,
E. A. Albanese, and B. L. Bachl.
1998.
Reproducibility of the Roche AMPLICOR polymerase chain reaction assay for detection of infection by Chlamydia trachomatis in endocervical specimens.
Clin. Chem.
44:1575-1578 |
| 7. | Peterson, E. M., V. Darrow, J. Blanding, S. Aarnaes, and L. M. De La Maza. 1997. Reproducibility problems with the AMPLICOR PCR Chlamydia trachomatis test. J. Clin. Microbiol. 35:957-959[Abstract]. |
| 8. |
Puolakkainen, M.,
E. Hiltunen-Back,
T. Reunala,
S. Suhonen,
P. Lahteenmaki,
M. Lehtinen, and J. Paavonen.
1998.
Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection.
J. Clin. Microbiol.
36:1489-1493 |
| 9. | Stary, A., S.-F. Ching, L. Teodorowicz, and H. Lee. 1997. Comparison of ligase chain reaction and culture for detection of Neisseria gonorrhoeae in genital and extragenital specimens. J. Clin. Microbiol. 35:239-242[Abstract]. |
Journal of Clinical Microbiology, June 2000, p. 2416-2418, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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