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Two commercial
nucleic acid amplification assays (PCR and LCR) for the detection of
C. trachomatis detected DNA in clinical urine samples with
equal efficiency in field testing.
External quality assessment program
for Chlamydia trachomatis diagnostic testing by nucleic acid
amplification assays.
Land S, Tabrizi S, Gust A, Johnson E,
Garland S, Dax EM.
J Clin Microbiol.
2002;40:2893-2896
Summary:
Question
What are the sensitivities of two commercial nucleic acid amplification
assays used widely in Australia for the detection of C. trachomatis
at the lower limit of detection and for detection in clinical urine
samples?
Design
This study describes the results of a voluntary external quality
assessment (EQA) program conducted in Australian diagnostic laboratories
testing for C. trachomatis using either the Amplicor Chlamydia
trachomatis test (Roche Diagnostic Systems, Inc., Branchburg, NJ) or
the LCx Chlamydia trachomatis assay (Abbott Laboratories, Abbott
Park, IL). The EQA program was used to compare the field performances of
the diagnostic assays.
Participants
Fifty-seven Australian diagnostic laboratories testing for C.
trachomatis participated.
Description of Tests and Diagnostic
Standard
The PCR assay (Roche Diagnostic Systems) and the LCR assay (Abbott
Laboratories) are qualitative and amplify a cryptic plasmid present in all
serovars of C. trachomatis. The assays were performed according to
the manufacturers' directions, using the recommended cut-off levels.
Results reported as equivocal were considered negative. Repeat testing was
not possible due to low sample volumes. The PCR assay included an internal
control of PCR amplification. After sample preparation, the detection
component of the PCR assay could be performed manually or automated using
the COBAS system (Roche). Results of the two methods of detection of the
PCR assay were analyzed together. Participating laboratories were
instructed to process panel samples as part of their routine testing
procedure.
Two EQA panels were evaluated. The first one, designed to contain low
levels of target DNA, was produced by spiking normal urine with sonicated C.
trachomatis grown in HeLa cells. The amount of C. trachomatis
added was determined by testing in quadruplicate serial ten-fold dilutions
of the sonicate at three reference laboratories. The end-point was defined
as the dilution at which 50% of the results were positive. The EQA panel
consisted of quadruplicate samples containing the endpoint concentration,
duplicate samples containing10 and 100 times the endpoint concentration,
and duplicate negative samples. The second panel consisted of five samples
in duplicate, four collected from individuals presenting with overt
symptomatic genital C. trachomatis infection, and one normal urine.
One positive sample was diluted 1:1000 in negative urine. Another positive
sample was spiked with humic acid to inhibit DNA amplification. The panel
was tested at three reference laboratories between 20 and 40 times. The
results were concordant within each assay for all replicates. Panels were
frozen and shipped on dry ice.
Main Outcome Measures
For the first panel, the detection rate for each assay was calculated,
counting each test as one result. For the second panel, the level of
concordance of duplicate samples with the reference laboratories' results
was determined.
Main Results
Fifty-one laboratories tested the first panel of samples. For all of the C.
trachomatis-spiked samples in the panel, the PCR assay showed a higher
level of detection than the LCR. For the samples containing the endpoint
concentration, 15 of 160 (9%) samples were C. trachomatis positive
by PCR compared to none of 44 by LCR (P < 0.05). However, there was no
significant difference in the detection rates of C. trachomatis DNA
between the PCR and the LCR assays for the samples containing 10 and 100
times the endpoint concentration.
Fifty-seven laboratories tested the
second panel. The level of concordance with reference laboratory results
is shown in the table. There was no significant interassay difference in
the levels of detection of C. trachomatis. Thirty-one of 47
laboratories using the PCR assay reported the presence of inhibitors in
the humic acid-spiked sample. Sixteen laboratories using PCR and all 10
laboratories using LCR found this sample negative because none of these
laboratories assayed for the presence of inhibitors.
| Concordance among 57
laboratories testing panel 2 samples |
| C.
trachomatis status |
Laboratory
concordance with reference results (%) |
PCR COBAS
(n = 33) |
PCR
manual
(n = 14) |
LCR
(n = 10) |
| Positive |
32 (97) |
13 (94) |
9 (90) |
| Positive |
22 (68) |
7 (50) |
9 (90) |
| Positive
(low level) |
21 (62) |
10 (71) |
9 (90) |
| Positive/Inhibitors |
23 (70) |
8 (57) |
0 |
| Negative |
32 (97) |
14 (100) |
10 (100) |
Authors' Conclusions
Both the PCR and the LCR assays evaluated in
this study detected C. trachomatis DNA in clinical urine samples
with equal efficacy in field testing. PCR demonstrated increased
sensitivity at lower C. trachomatis concentrations compared to LCR.
Source of funding: The Diagnostic and
Technology Branch of the Australian Government Department of Health and
Aged Care.
For correspondence: Sally Lund,
National Serology Reference Laboratory, St Vincent's Institute of Medical
Research, 41 Victoria Parade, Fitzroy, Victoria, Australia. E-mail
address: sally@nrl.gov.au.
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