Chlamydia trachomatis, Neisseria gonorrheae (CT/GC) NAT

The Aptima Combo 2® Assay is a target amplification nucleic acid probe test that utilizes target capture for the in vitro qualitative detection and differentiation of ribosomal RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal disease using the Panther® System as specified.  On the Panther System, the assay may be used to test the following specimens from symptomatic and asymptomatic individuals: clinician-collected endocervical, vaginal, throat, rectal, and male urethral swab specimens, clinician-collected gynecological specimens collected
in the PreservCyt® Solution, patient-collected vaginal swab specimens,1 and female and male urine specimens.

About Chlamydia trachomatis, Neisseria gonorrheae

Chlamydia trachomatis and Neisseria gonorrhoeae infections are the most common sexually transmitted bacterial diseases in the United States. Approximately 4 million new chlamydia cases are estimated to occur each year in the United States with worldwide estimates of approximately 50 million new cases annually.^1-3 The incidence of chlamydial infections in women in the US in 1996 was 186.6 per 100,000. The total number of chlamydial infections and gonorrhea cases reported in the US in 1996 were 490,080 and 325,883, respectively.²

Chlamydiae are gram-negative, obligate intracellular bacteria. They form characteristic intracellular inclusions which can be observed in cell culture by light microscopy after special staining is applied.⁴ Chlamydia trachomatis causes cervicitis, urethritis, salpingitis, proctitis and endometritis in women and urethritis, epididymitis and proctitis in men. Acute infections are reported more frequently in men because women often have no symptoms of infection. It has been estimated that 70 - 80% of women and up to 50% of men who are infected experience no symptoms. Many chlamydial infections in women remain untreated which may result in low-grade inflammation in the Fallopian tubes, a leading contributor to infertility. This organism can also be transmitted in the birth canal, potentially resulting in infant conjunctivitis and/or chlamydial pneumonia in newborns.^4,5

Neisseria gonorrhoeae are gram-negative, oxidase positive diplococci which can be observed in Gram-stained smears of urethral discharges, usually within neutrophils. Culture of N. gonorrhoeae can be difficult because the organism does not survive long outside its host and is highly susceptible to adverse environmental conditions such a drying and extreme temperatures.⁶ Neisseria gonorrhoeae causes acute urethritis in males, which if untreated can develop into epididymitis prostatitis, and urethral stricture. In females, the primary site of infection is the endocervix. An important complication in females is development of pelvic inflammatory disease which contributes to infertility.⁷ Asymptomatic infections occur often in females but infrequently in males.

The current methods for detection of C. trachomatis and/or N. gonorrhoeae include culture, immunoassays, non-amplified probes, and amplified probes.^4,6,7 The development of amplified methods has demonstrated two advantages over non-amplified methods: increased sensitivity, and applicability to a variety of sample types. Historically, culture has been the "gold standard" for detection of C. trachomatis. However, the culture yield varies widely among laboratories, and culture in routine practice is less sensitive than amplified methods. Combining results from multiple methods of CT detection improves accuracy for evaluating new tests in that infected and uninfected patients can be more reliably identified. For identification of GC, optimized culture methods continue to be the standard for diagnosing patients with gonococcal infections.

The Aptima Combo 2 Assay combines the technologies of target capture, TMA, and DKA.

Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the rRNA targets and protect them from degradation during storage. When the Aptima Combo 2 Assay is performed in the laboratory, the target rRNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence
specific regions of the capture oligomers bind to specific regions of the target molecules. The capture oligomer:target complex is then captured out of solution by decreasing the temperature of the reaction to room temperature. This temperature reduction allows hybridization to occur between the deoxyadenosine region on the capture oligomer and the poly-deoxythymidine molecules that are covalently attached to the magnetic particles. The microparticles, including the captured target molecules bound to them, are pulled to the side of the reaction vessel using magnets and the supernatant is aspirated. The particles are washed to remove residual
specimen matrix that may contain amplification reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.  Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima Combo 2 Assay replicates a specific region of the 23S rRNA from CT and a specific region of the 16S rRNA from GC via DNA intermediates. A unique set of primers is used for each target molecule. Detection of the rRNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent DNA probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The labeled DNA probes combine with amplicon to form stable RNA:DNA hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals in a luminometer, and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the CT and GC labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection reaction for CT signal has very rapid kinetics and has the “flasher” kinetic type. The chemiluminescent detection reaction for GC signal is relatively slower and has the “glower” kinetic type. Assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

Test performed by Eurofins DPT, 6933 S. Revere Parkway, Centennial, CO 80112. This test has been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.