PAP/NGC Programs Review
Marianne Prey, MD
Ann Moriarty, MD
Epidemiology | Biology |
Screening recommendations | Testing
methods | ThinPrep requirements | SurePath
requirements | Constraints
With the advent of liquid-based cytology, cells are collected and maintained
in liquid preservative that were never available with conventional Pap smears.
Human papillomavirus testing was the first “out-of-the-vial” test
performed, but because of the availability of preserved cells, the door has
been opened for myriad other tests as long as they do not require live cells
or living organisms. Thanks to nucleic acid hybridization tests, tests can be
developed that can be piggybacked on the routine Pap test. Ancillary out-of-the-vial
testing makes the most sense if the tests are a screening test, like the Pap
test, and intervention at an early stage can decrease morbidity. Chlamydia
trachomatis is a common “silent” infection of women in the
United States, and one that causes significant health problems. C. trachomatis
testing out of the vial makes perfect sense in a Pap screening program.
Epidemiology. C. trachomatis
is considered the most common cause of sexually transmitted disease in the United
States. Centers for Disease Control and Prevention statistics indicate that
the incidence is steadily increasing, with an estimated 3 million new cases
each year. Five percent to 15 percent of reproductive age women are believed
to be infected. Teenage girls age 15 to 19 represent 46 percent of infections,
and women age 20 to 24 represent 33 percent of infections. Left untreated, 40
percent of infections will result in pelvic inflammatory disease, or PID. Of
those women with PID, one in five will become infertile, one in five will develop
chronic pelvic pain, and one in 10 will have an ectopic pregnancy. In addition,
women infected with C. trachomatis have a three to five times increased
risk of acquiring HIV and a 50 percent chance of transmitting infection to their
infant during childbirth. The majority of women infected with C. trachomatis
are unaware of their infection because symptoms are absent or mild and, therefore,
women do not seek medical attention. Overall, $1.7 billion is spent annually
in the U.S. to treat Chlamydia infections.
Biology. C. trachomatis is
an obligate intracellular parasite with tropism for columnar epithelial cells
though there is no known receptor. There are two morphologic forms of the C.
trachomatis organism: the infectious form that is the elementary body and
the metabolically active form that is the reticulate body. The elementary body
contains ribosomal RNA and has limited extracellular survival. The reticulate
body has a circular DNA genome and exists as an intracellular organism only.
The organism gains entrance to the host cell by attachment of the elementary
body to endocervical cell microvilli and engulfment in an endosome. It transitions
to a reticulate body, wherein DNA, RNA, and protein synthesis occur followed
by binary fission. The reticulate body uses the host ATP and depletes the endocervical
cell of its nutrients as it matures into an elementary body. After 72 to 96
hours of inclusion, the elementary bodies are extruded and available to infect
C. trachomatis will cause acute mucopurulent endocervicitis. Patients
who are pregnant and those who have an ectropion of the cervix are at increased
risk for acquiring infection. Only one-third of infected patients are symptomatic,
and these symptoms may include “acute urethral syndrome” due to
urethral and bladder involvement and proctitis due to rectal involvement. In
addition, C. trachomatis infections are associated with other bacterial
infections, primarily gonorrhea. Women with C. trachomatis infection
have an abnormal colposcopic examination associated with follicular cervicitis,
inflammatory exudate, and atypia of squamous, endocervical, and metaplastic
Because C. trachomatis is an enormous public health concern, several
organizations have put forth recommendations for routine screening. The U.S.
Preventive Services Task Force recommends that all sexually active women age
25 and younger and asymptomatic women at risk be screened routinely. CDC guidelines
call for Chlamydia screening at least annually for all sexually active
females under age 20, annually for women over age 20 if at risk, all women with
cervicitis, and all pregnant women. Health Plan Employer Data and Information
Set, or HEDIS, requires that sexually active women be screened for Chlamydia.
A woman is considered sexually active if she is seeking care for any of the
following: pregnancy, contraception, sexual assault or abuse, history of sexually
transmitted diseases, or gynecological services. To minimize missed screening
opportunities, HEDIS recommends that all sexually active women age 15 to 25
be screened. All 50 states require reporting of positive Chlamydia
Testing methods. Specimens for
C. trachomatis testing must be collected directly from mucous membranes.
Mucopurulent discharge alone is not an appropriate specimen. Since wood, calcium
alginate, and occasionally cotton is toxic to the organism, specimens must be
collected using a Dacron or rayon-tipped swab with a plastic or nonaluminum
Culture is considered the reference method for C. trachomatis, but
material collected into liquid-based Pap transport media is unsuitable for culture.
The Cobas Amplicor CT/NG test is FDA approved for testing endocervical swab,
male and female urethral swab, male and female voided urine, and ThinPrep Pap
test specimens. This test uses PCR amplification of target DNA followed by hybridization
of the amplified DNA to oligonucleotide probes. Detection of the probe-bound
amplified DNA is by colorimetric determination. Thirty-five cycles of amplification
increase the DNA from one copy to more than 1 billion. This process results
in a negative predictive value of 98 percent to 99 percent and a positive predictive
value of 21 percent to 87 percent depending on the prevalence of disease in
the population. False-negative results are mostly due to competitive and noncompetitive
Competitive inhibition occurs when one organism is present in relatively high
concentrations as compared with the internal control. In the Cobas Amplicor
CT/NG test, all three DNA targets (C. trachomatis, Neisseria gonorrhoeae,
and internal control) compete for primers and nucleotides. The reactants are
consumed by the amplification of the organism that is present in high concentration,
resulting in no amplification for the control and for the other organism. In
this situation, it is not possible to determine if the second organism is absent
from the sample or if the negative result is due to competitive inhibition.
In noncompetitive inhibition there is something in the patient’s sample
that interferes with the amplification process. Mucus and lubricant are considered
to be common causes of noncompetitive inhibition.
The most common false-positive interfering substance is blood when it is present
at a concentration of greater than five percent (v/v). No cross-reactivity has
been identified for 132 bacteria, six fungi, one protozoan, and 11 viral strains
commonly isolated from the urogenital tract. This includes Candida,
Trichomonas, HPV, and all of the bacteria composing bacterial vaginosis.
Contamination may cause false-positives; laboratories performing nucleic acid
testing must be aware of the risk of cross-contamination causing false-positives
and follow meticulous methods to avoid cross-contamination if using liquid-based
The PreservCyt Solution component of the ThinPrep 2000 system may be used as
an alternative collection and transport medium for gynecologic specimens tested
with the Roche Cobas Amplicor CT/NG test. However, additional specimen handling
procedures are required according to the product insert (Cytyc Corp., 85 Swanson
Road, Boxborough, MA 01719). “Pre-aliquoting” or “pre-quot”
testing is not FDA approved at this time for C. trachomatis testing
from liquid-based Pap media.
The process begins with the patient’s gynecologic sample collected by
the clinician using either a broom-type collection device or a cytobrush and
spatula in combination. The sampling device(s) is rinsed in a vial of PreservCyt
Solution, which is then capped, labeled, and sent to a laboratory for a ThinPrep
After processing for the ThinPrep Pap test, the PreservCyt sample vial is recapped
and sent for additional C. trachomatis and N. gonorrhoeae
testing using the Roche Diagnostics Cobas Amplicor CT/NG test.
The following are the procedural steps that are required to prevent cross-contamination
during preparation of the ThinPrep Pap test slide when subsequent CT/NG testing
- Glove hands
- Using a 10 percent (volume/volume) bleach bath, soak two filter caps. (Note:
The use of two filter caps allows for one to be soaked while the other is
in use.) Always ensure that a filter cap has soaked a minimum of one minute
before using for sample processing.
- Remove one filter cap and dip three times in distilled water. Dry thoroughly.
- Place onto a BloodBloc Super Absorbent Wipe, and lubricate O-rings as necessary.
- Change gloves (if O-rings were lubricated).
- Load the fixative bath into place on the ThinPrep 2000 Processor.
- Using a new TransCyt Filter, insert the filter into the prepared filter
cap. (These components together are called the filter assembly.)
- Insert the filter assembly into the ThinPrep 2000 Processor.
- Load the PreservCyt sample vial; ensure that when removing the cap from
the PreservCyt sample vial, it is placed with threads facing upward on the
- Load the ThinPrep microscope slide.
- Close door and initiate gyn sequence No. 4.
- Upon completion of slide prep aration, open door, remove PreservCyt sample
vial and fixative bath containing prepared slide. Remove slide and place it
in a bath of 95 percent ETOH. Return fixative bath to processor.
- Remove filter assembly using a BloodBloc Super Absorbent Wipe. Separate
filter from filter cap, and discard the filter together with the BloodBloc
Super Absorbent Wipe.
- Return filter cap to bleach bath.
- Change gloves. Repeat steps two through 14 for slide processing on each
subsequent sample processed on the ThinPrep 2000 Processor.
The Roche Diagnostics Cobas Amplicor CT/NG test cannot be used on samples processed
using the ThinPrep 3000 Processor. PreservCyt Solution used as an alternative
collection and transport medium with the Roche Cobas Amplicor CT/NG test is
for use only with cervical specimens.
Roche Diagnostics Cobas Amplicor CT/NG test is currently not FDA approved for
testing of cervical specimens that have been collected in a SurePath vial. If
a laboratory uses the method off label, internal validation must be performed
and rigorous methods to prevent cross-contamination must be implemented. The
following additional procedural steps to the SurePath slide preparation process
have been followed in some laboratories to validate the method without cross-contamination:
- Glove hands
- Dip Prepmate rack in 10 percent bleach and dry before loading samples.
- Always work from clean to dirty when loading racks.
- Once the Prepmate processing is complete, wipe the top of the SurePath vials
with an alcohol prep pad or gauze dipped in 70-95 percent alcohol. Then replace
the punctured cap with a new unpunctured cap. Remove the punctured cap by
holding the cap with a plastic-backed biowipe. Discard the cap and the biowipe
in one motion.
- Change gloves between each Prepmate rack.
- Trays or containers that are used for transportation of the SurePath vials
from the cytology department to the CT/NG testing department should be cleaned
with 10 percent bleach before adding the vials.
Constraints. The use of C. trachomatis
testing from liquid-based Pap transport media is sensible. C. trachomatis
infection is widespread in the population that receives Pap tests and is associated
with significant morbidity. Recommended guidelines for screening involve young
women of childbearing age, who are usually in screening programs for cervical
carcinoma. What are the constraints?
First, C. trachomatis screening, like cervical cancer screening, is
a screening test with false-positives and false-negatives. C. trachomatis
testing off the vial is not a culture, and practitioners must be educated about
the causes of false-negative and false-positive tests.
Second, present FDA approved C. trachomatis out-of-vial testing is
performed after the Pap test and therefore requires careful methods to avoid
cross-contamination in preparation of the specimen. Aliquotting before the Pap
test is performed is considered off-label use and must be validated within the
Third, the ThinPrep Pap test PreservCyt Solution is the only media approved
now for nucleic acid testing. Internal lab validation of SurePath collection
media must be performed before using these liquid-based Pap specimens.
Finally, though liquid-based Pap tests provide abundant cells for analysis,
it is not an inexhaustible supply of nucleic acid. The collection of cells still
depends on the clinician’s procurement technique, the ability to transfer
cells from the collection device to the media, and the transfer of the nucleic
acid into the test system. Cells must be collected in adequate amounts for screening
for cancer and HPV first and foremost. If labs routinely pre-aliquot specimens
for other tests, the effectiveness of the liquid-based Pap test to detect cervical
cancer may be hindered. The value of the additional tests must be weighed against
the “dilution” of the Pap test to detect cervical cancer.
Dr. Prey is laboratory medical director, Quest Diagnostics, St. Louis, Mo.
Dr. Moriarty is with AmeriPath-Indiana, Indianapolis. Both are members of the
CAP Cytopathology Committee.