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