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An in-house sample
preparation procedure for endocervical swabs improved the
sensitivity of the C. trachomatis Cobas Amplicor assay.
Improved
sensitivity of the Chlamydia trachomatis Cobas Amplicor using an
optimized procedure for preparation of specimens.
Niederhauser C, Kaempf L.
Eur J Clin Microbiol. 2003;22:118-121.
Summary:
Question
Can a change in the recommended sample preparation procedure for
endocervical swab specimens increase the sensitivity of the C.
trachomatis Cobas Amplicor assay?
Design
A modified sample preparation procedure for the Cobas Amplicor Chlamydia
trachomatis assay that increased the initial sample volume,
concentrated the sample by centrifugation, and digested the sample with
protease was compared to the manufacturer's recommended protocol for the
detection of C. trachomatis DNA in endocervical swab samples.
Participants
Five hundred sixteen women attending a family planning clinic for a
routine check-up during pregnancy, abdominal pain, premature contractions,
vaginal discharge, etc., and those with suspected adnexitis or cervicitis
were tested.
Description of Tests and Diagnostic
Standard
Endocervical swab samples were extracted by two methods. The first
extraction was performed according to the instructions provided by the
manufacturer of the Cobas Amplicor Chlamydia trachomatis assay
(Roche, Switzerland), resulting in 1/160 of the original sample being
added to the PCR reaction. In the second extraction, the swabs were
collected in 2 ml of 2-SP medium and centrifuged. The cell pellets were
washed 2 times, suspended in 0.1 ml of 1:1 2-SP medium/lysis buffer
(Roche), incubated for 10 min at room temperature, and digested with 0.1
ml of Proteinase K solution. Fifty microliters, representing ¼ of the
original sample, was added to the PCR reaction. Nine serial four-fold
dilutions of three C. trachomatis positive samples were prepared
and extracted by both methods. Nine serial four-fold dilutions of one
bloody endocervical sample and a 2-SP medium control spiked with C.
trachomatis were extracted using the Roche method and the modified
method that was further modified by suspending the cell pellet obtained
after the first centrifugation in 1 ml of erythrocyte lysis buffer (Argene/Biosoft,
France), then processing as described above. After processing, all samples
were tested using the Cobas Amplicor Chlamydia trachomatis assay
(Roche) according to the manufacturer's instructions.
Main Outcome Measures
The C. trachomatis PCR amplification results of samples extracted
using the modified method were compared to the results of duplicate
samples prepared according to the Cobas Amplicor instructions.
Main Results
The PCR amplification results of the 4-fold dilution series of three C.
trachomatis positive samples showed that samples extracted with the
modified procedure were positive in two to three dilution steps further
than those extracted using the recommended assay method. Of 516
endocervical swab samples tested after extraction by both methods, 8
samples were positive by both methods, and 7 were positive only with the
modified method. Only 4 (0.87%) of 516 samples were inhibited when the
original Roche method was used compared to 39 (7.56%) when the modified
protocol was used. Thirty-one of the 39 inhibited samples were
contaminated with blood. However, no difference was seen in the PCR
results from the dilution series of the blood-contaminated clinical sample
analyzed with the Roche procedure compared with the sample analyzed with
the modified procedure.
Authors' Conclusions
The modified procedure is labor-intensive
and not suitable for large-scale screening. However, the improved protocol
will result in detection of low-level infections, allowing earlier
therapeutic interventions.
Source of funding:
Roche Diagnostics
For correspondence: Christoph
Niederhauser, Blood Transfusion Service Bern, Swiss Red Cross,
Murtenstrasse 133, 3008 Bern, Switzerland. E-mail address:
christoph.niederhauser@bsd-be.ch.
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