Detection of Chlamydia trachomatis (CT)
& Neisseria gonorrhoeae(GC) by APTIMA
COMBO 2 ASSAY
Molecular Diagnostics
Instructor: Dr. Nancy McQueen
Date June 3rd, 2004
Stacey Stubblefield & Anahid Mirzatoni
Urogenital Diseases
• Chlamydia trachomatis(CT)& Nesseria
gonorrhoeae(GC) infections are both caused by
non-motile gram –negative bacteria.
• (CT) &(GC) are the two most common sexually
transmitted infections worldwide & in the U.S.
• Half of the patients are usually coinfected with
(CT) & (GC).So, it is imperative to test for both in
sexually active individuals.
• The infections occur in both male & females (46%
of (CT) occurs in females). The infected
individuals could be symptomatic or
asymptomatic.
Urogenital Disease cont,
• (CT) can cause urethritis, epididymitis,
proctitis, cervicitis, acute salpingitis &
Pelvic Inflammatory Disease(PID).
• (GC) is the causitive agent of gonorrheal
disease and can cause (PID) that manifest as
endometritis, salpingitis, pelvic peritonotis
& tubo-ovarian abscesses.
APTIMA COMBO 2 ASSAY
• It is a second generation nucleic acid
amplification test that is used for in vitro detection
& differentiation of rRNA from CT & GC.
• AC2 assay is a qualitative molecular diagnostic
assay that uses a family of Gen-Probe’s proven
technologies including target capture (TC),
Transcription-Mediated Amplification (TMA) and
Dual Kinetic Assay(DKA).
• Since it provides qualitative results, there is no
correlation between the magnitude of a positive
assay signal & the number of organisms in a
specimen.
Different Detection Techniques For (CT) & (GC)
1.
2.
3.
4.
Cell Culture
Enzyme immunoassay(EI)
Direct antibody testing
Nucleic acid amplification test(NAAT)
DNA probe assay
5. Direct probe assays
Cell Culture Technique Advantages:
• It is specific & considered to be the “gold
standard” for (CT) detection.
• Organism is first grown on the selective media &
then its nucleic acid is detected by DNA probe.
Cell Culture Technique Disadvantages:
• It is highly dependent on proper handling, storage,
& transport of specimens, that could result in the
loss of organism viability& yield false negative
results if not handling efficiently.
• According to scientific publications, it has less
clinical sensitivity compared to NAAT technique.
NAAT Technique Advantages:
• It consistently exceeded the sensitivities of NONNAAT methods( cell culture , direct probe, EIA).
• The ability to produce a positive signal from a
single copy of target DNA or RNA.
• Detection ability of NAAT without a pelvic
examination or intrauretheral swab specimen (for
males) & testing urine.
• CDC recommends use of NAAT to screen for
genitourinary infections with CT or GC.
NAAT Technique Disadvantages:
• It is highly expensive.
• Specimen can contain amplification inhibitors that
result in false-negative (decreasing specificity).
• Cross-reactivity of primers used for N. gonorrhea
detection with non-gonococal Neisseria species.
• More susceptible than non-NAAT methods to false
positive results due to contamination, if control
procedure is not applied accurately.
EIA Technique Advantages:
It is rapid, simple and reproductible
No sample pretreatment is needed
EIA Technique Disadvantages:
• Cross- reactivity of AB with other
microorganism including other Chlamedia
species.(Decreasing specificity and
increasing the chance of having false
positive)
• Lack of accuracy to detect more than one
organism.(Less sensitivity)
Direct Probe Technique Advantages:
• It is highly specific, because of the direct
detection of organism nucleic acid in specimen &
lack of cross-reaction with other targets.
Direct Probe Technique Disadvantages:
• It is not very sensitive for organisms such as
viruses that exist in too few numbers to be directly
detected with DNA probe.
Why Do We Use APTIMA COMBO 2
ASSAY?
• High sensitivity ( it is the only assay with urine
specimen sensitivity equivalent to swab specimen.
• It is cost- effective by providing two results from
one sample.
• Increasing laboratory efficiency with single tubetesting.
• Eliminating inhibition problems & crossreactivity.
Sample Preparation & Purification of Target
Molecules by TC Method
• Collection of swab or urine specimen into
transport tubes respectively. Transport solution
releases rRNA targets & protects them from
degradation.
• Isolation of target rRNA from urine & swab
samples by the usage of capture oligomers in
target capture.
• The capture oligomers contain a string of
deoxyadenosine residues & sequences
complementary to specific regions of the target
molecules.
• A separate capture oligomers is used for each
target.
• Binding of the sequence specific
regions of the capture oligomers to
the target molecule.
• Temperature reduction causes
hybridization to occur between the
deoxyadenosine region on the
capture oligomer and the polydeoxythymidine molecules
(attached covalently to magnetic
particles).
• The magnetic particles are pulled to
the side of the tube by a magnet and
specimen matrix that may contain
amplification reaction inhibitors are
washed.
• After Target Capture step is
completed, the specimens are ready
for amplification.
Advantages of Target Capture
• Eliminates centrifugation and chemical extraction
steps.
• Allows simultaneous targeting of multiple nucleic
acid sequences.
• Allows the use of large volumes and a variety of
samples that are currently difficult to amplify such
as cerebral spinal fluid and serum.
• Increases specificity, because target nucleic acids
are purified before amplification.
• Increases sensitivity by reducing the inhibition
rate.
Transcription Mediated Amplification Assay
(TMA)
• Using specific primers for each target nucleic acid
strands.
• Reaction replicates a specific region of the 23S
rRNA from chlamydia trachomatis (CT) and a
specific region of the 16S rRNA from Neisseria
gonorrhoeae (GC) via DNA intermediates.
• Detection of the rRNA amplification product
sequence (amplicon) is achieved using nucleic
acid hybridization.
Target Amplification Procedure
TMA cont,
•One primer contains a T7 promoter sequence for RNA
polymerase that hybridizes to the target RNA
•RT creates a cDNA of the target RNA by extension from
the 3’ end of the promoter-primer
•The RNA of the resulting RNA:DNA duplex is degraded
by the RNAse H activities of the RT
•A second primer then binds to the cDNA containing the
promoter sequence from the T7 promoter-primer
Advantages of Transcription-Mediated Amplification Assay
• TMA produces 1001000 copies per
cycle which results
in a 10 billion fold
increase of copies
within about 15-30
minutes
• TMA produces RNA
amplicon rather than
DNA amplicon.
• TMA is highly
specific.
Amplicon Detection by Hybridization
Protection Assay (HPA)
• Single-stranded chemiluminescent DNA probes, are
labeled with different acridinium ester molecules and are
complementary to a region of each target amplicon.
• Stable RNA:DNA hybrids are then formed from the
labeled DNA probes binding to amplicon.
• The Selection Reagent eliminates the generation of signal
from unhybridized probe by differentiating hybridized
from unhybridized probe.
• In the detection step, light emitted from the labeled
RNA:DNA hybrids is measured as photon signals and are
reported as Relative Light Units (RLU) in a luminometer.
Dual Kinetic Assay (DKA)
• In DKA, Chlamydia trachomatis and
Neiseria gonorrhoeae labeled probes have
differences in their kinetic profiles that
allow for the differentiation of signal.
• Kinetic Profiles are derived from
measurements of photon output during the
detection read time.
Dual Kinetic Assay (DKA)
• One of the acridinium ester molecules attached to one of
the DNA probes has fast light-off kinetics and is called a
“flasher”.
• The other acridinium ester molecules attached to other
DNA probe that has slower kinetics is called a “glower”.
• The chemiluminescent detection reaction for CT signal has
rapid kinetics and has the “flasher” kinetic type, while the
chemiluminescent detection reaction for the GC signal is
slower and has the “glower” kinetic type.
• Cut-off based on the total RLU and the kinetic curve type
is how the assay results are determined.
Advantages of Dual Kinetic Assay (DKA)
• Allows for the simultaneous detection of
amplicons from two independent
amplification reactions (multiplex
amplification).
Controls
• Control
Total RLU
+C CT/ -C GC
+C GC/-C CT
> 100 and < 3000
> 150 and < 3000
CT Result
CT positive
CT negative
GC Result
GC negative
GC positive
• A negative result does not preclude a possible infection because results are
dependent on adequate specimen collection, absence of inhibitors, and
sufficient rRNA to be detected.
• Low positive control values may be caused by incorrect temperatures or time
selection during various steps.
• Test results may be affected by:
• Technical error
• Specimen mix-up
• Improper specimen storage
Limitation of APTIMA Combo 2 Assay:
• Therapeutic failure or success cannot be
determined , since nucleic acid may persist after
antimicrobial therapy.
• It is not a quantitative assay.
• This assay does not permit microscopic
assessment of specimen adequacy, so training of
the techniques is necessary.
Additional Applications of APTIMA
COMBO 2 ASSAY
• Highly sensitive multiplex assay for
detection of HIV- type1 virus & HC virus.
• Detection of HB virus DNA/ HC virus
RNA/ HIV-type1RNA.
• TMA based assay for detection of West Nile
virus in blood, plasma, & organ donors
specimen
REFERENCES
• Gen-Probe (2000). New Directions in Molecular Diagnostic Testing
Pioneer advances in Health Care. CA.pp3-12.
• Arup (2004). Chlamydia trachomatis and Neisseria gonorrhoeae by
Amplified Detection (APTIMA). CA. pp1-3.
http://www.aruplab.com/guides/clt/tests/clt_a141.jsp
• OML Laboratories (2002) Chlamydia trachomatis and Neisseria
gonorrhoeae, TMA Amplified by Gen-Probe APTIMA Combo 2 Assay
Eugene,OR. Pp.1-2 http://www.omlabs.com
• Center For Disease Control (2002). Screening Tests to Detect
Chlamydia trachomatis and Neiseria gonorhoeae Infections pp. 2-6
http://cdc.gov/mmwr/preview/mmwrhtml/rr5115a1.htm
• Fadiman, K., Goldman, S. (2004). Screening for Chlamydia
trachomatis American College of Preventive Medicine. San Francisco,
CA. pp. 1-3