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PreservCyt could be
used as an alternative medium for the collection and transport of
specimens for the detection of C. trachomatis and N.
gonorrhoeae by ligase chain reaction.
Comparison of
methods for detection of Chlamydia trachomatis and Neisseria
gonorrhoeae using commercially available nucleic acid
amplification tests and a liquid Pap smear medium.
Koumans EH, Black CM, Markowitz LE,
Unger ER, Pierce A, Sawyer MK, Papp JR.
Journal of Clinical
Microbiology 2003;41:1507-1511.
Summary:
Question
What is the stability of chlamydial and gonococcal DNA in PreservCyt, a
methanol-based liquid cytology medium? How well do the results of a ligase
chain reaction (LCR) assay performed on cervical samples placed in
PreservCyt agree with those of samples placed in LCx medium? What is the
performance of the LCR assay on cervical samples in PreservCyt compared to
NAATs and culture performed on conventional samples for the detection of C.
trachomatis and N. gonorrhoeae infections?
Design
This study describes 1) an evaluation of the stability of C.
trachomatis and N. gonorrhoeae DNA in PreservCyt, 2) the
agreement between the results of cervical samples tested by LCR when
placed in PreservCyt compared to LCx medium, and 3) a blinded comparison
of the performance of the LCR assay on cervical samples in PreservCyt with
those of culture, PCR, and TMA on samples from multiple sites for the
detection of C. trachomatis and N. gonorrhoeae in adolescent
women.
Participants
A subset of 225 nonpregnant, HIV-negative, sexually active adolescent
women, aged 12 to 19 years old (mean = 16.6 years) attending an adolescent
clinic at a public pediatric hospital in Atlanta, GA, who were part of a
larger longitudinal STD study, and who required a pelvic exam were tested.
Women who had received antibiotics in the previous month were excluded.
Description of Tests and Diagnostic
Standard
For the assessment of DNA stability in PreservCyt, three different
concentrations of C. trachomatis (1000, 100, and 10
inclusion-forming units/ml) and 3 different concentrations of N.
gonorrhoeae (1000, 100, and 10 CFU/ml) were added to three aliquots
each of PreservCyt (Cytyc Corp., Boxborough, MA) that were incubated at
room temperature, 4oC, and 37oC and tested after 24 h, 48 h, and 7 d,
producing a total of 27 samples for each organism. Dacron swabs saturated
in liquid from each concentration were immersed in PreservCyt and tested
under the same conditions.
Clinical samples collected from each
woman included separate endocervical swabs for N. gonorrhoeae and C.
trachomatis detection by culture, PCR (COBAS Amplicor, Roche
Diagnostic Systems, Branchburg, NJ), LCR (LCx, Abbott Laboratories,
Chicago, IL), and TMA (Amp CT, Gen-Probe, Inc., San Diego, CA) (C.
trachomatis only), and a cervical broom placed in PreservCyt, a vaginal
swab, and urine for N. gonorrhoeae and C. trachomatis
detection by LCR, PCR, and both, respectively. The swab for N.
gonorrhoeae culture was collected first, the broom sample second, and
the others in random order.
N. gonorrhoeae was cultured on
Martin-Lewis agar plates, an oxidase test and Gram staining were performed
on suspected colonies after 24 and 48 h, and the NHI card or the rapid NH
strip (bio-Merieux Vitek, Hazelwood, MO) was used to confirm detection. C.
trachomatis was cultured on BGMK cells grown on coverslips in 1-dram
shell vials with 200 μl of inoculum and detected following 48 h of
incubation by staining with a fluorescein-labeled anti-MOMP monoclonal
antibody (Wampole Laboratories, Cranbury, NJ). Swab and urine specimens
for the NAATs were collected and tested according to the directions
provided by the manufacturers' package inserts. One ml of each PreservCyt
specimen was centrifuged, the pellet suspended in 1 ml of LCx urine
resuspension buffer, heated at 95 to 100oC for 15 min, and tested
according to the manufacturer's protocol.
Main Outcome Measures
Agreement and the kappa statistic were calculated to ascertain agreement
between the two cervical specimen collection methods (PreservCyt and LCx)
using the same assay (LCR). The sensitivity and specificity of the LCR
assay performed on cervical samples in PreservCyt were determined using a
patient reference standard based on the combined PCR results of cervical,
vaginal, and urine specimens, and a multitest reference standard based on
the combined results of culture, TMA, and PCR at any site.
Main Results
In the stability experiments, C. trachomatis and N. gonorrhoeae were
detected by LCR in all 27 spiked samples and swabs. The agreement between
LCR on cervical samples in LCx medium and in PreservCyt was 0.97 and 0.96,
and the kappa was 0.92 and 0.96 for detection of C. trachomatis and
N. gonorrhoeae, respectively. Of five C. trachomatis LCx
positive, PreservCyt negative subjects, all were positive by at least one
other test. Of three C. trachomatis PreservCyt positive, LCx
negative subjects, all were negative by all other tests.
Seventy (27%) of 255 subjects had
chlamydial infections and 27 (11%) had gonococcal infections by cervical
PCR. The performance of the LCR on cervical samples in PreservCyt compared
to a PCR patient standard (cervical, vaginal, and urine samples) and to a
multitest standard (culture, TMA, and PCR at all sites) for the detection
of C. trachomatis and N. gonorrhoeae is shown in the table.
Authors' Conclusions
PreservCyt medium is an acceptable specimen
type for the detection of C. trachomatis and N. gonorrhoeae by
LCR. A single cervical specimen can be used for cervical cytology and STD
screening using a nucleic acid amplification assay.
Source of funding: Cytyc Corporation,
Roche Diagnostics Corporation, and Gen-Probe Inc.
For correspondence: Emilia H. Koumans,
DSTD/NCHSTP, 1600 Clifton Rd. NE, MS E-02, Atlanta, GA 30329. E-mail
address: exk0@cdc.gov.
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