Evaluation of Chlamydia trachomatis and Neisseria gonorrhoeae Detection in Urine, Endocervical, and Vaginal Specimens by a Multiplexed Isothermal Thermophilic Helicase-Dependent Amplification (tHDA) Assay

Abstract

We have developed a new research assay that combines sequence-specific sample preparation and isothermal amplification for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae infections. The assay targets both the omp gene and the cryptic plasmid of C. trachomatis and the multicopy opa gene of N. gonorrhoeae, which are amplified and detected in a single reaction. We evaluated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in first-catch urine, swab, and liquid-based cytology samples. Total agreement between the new assay and APTIMA Combo 2 varied between 95.3% and 100%, depending on the sample type and target detected. Total agreement between the new assay and BD ProbeTec varied between 96.7% and 100%, depending on the sample type and target detected. The assay has a simple work flow, and endpoint results can be achieved in 3 h, including sample preparation. The assay described here was evaluated for research use and was compared to commercially available assays.Chlamydia trachomatis and Neisseria gonorrhoeae are among the most common causes of sexually transmitted infections (3). Infection with these organisms is often asymptomatic, which hinders diagnosis or treatment. Persistent infection can have serious negative consequences, including pelvic inflammatory disease and infertility. Untreated and undiagnosed infections also contribute to the further spread of the disease. Screening programs are recommended to prevent these consequences (17). Of the various testing methods currently available, nucleic acid amplification tests are among the most sensitive and specific methods for detection of C. trachomatis and N. gonorrhoeae organisms in clinical specimens (7, 9). The high sensitivity of nucleic acid amplification tests allows them to be used with less-invasive clinical samples, such as first-catch urine, which facilitates establishment of screening programs.The assay described here combines sequence-specific sample preparation and isothermal amplification for the detection of C. trachomatis and N. gonorrhoeae infections. Sequence-specific sample preparation is based on antibody capture of DNA-RNA hybrids. Ribo-oligonucleotide probes hybridize to specific target sequences, and the resulting hybrids are captured by antibodies specific for DNA-RNA hybrids. This process enriches the desired nucleic acid sequences in the sample. Antibodies are coupled to magnetic beads. Target amplification is accomplished by thermophilic helicase-dependent amplification (tHDA) (16). Amplification of captured strands of the DNA target occurs directly on magnetic beads through the combined action of primers, thermostable DNA polymerase, and helicase. Target DNA is detected by use of dually labeled probes in either endpoint or real-time detection modes. C. trachomatis and N. gonorrhoeae targets are amplified and detected simultaneously in a single closed reaction vessel.We investigated the ability of the assay to detect C. trachomatis and N. gonorrhoeae infections in clinical specimens. R...

Assessment of best single sample for finding chlamydia in women with and without symptoms: a diagnostic test study

“…We have identified 11 published papers where there are comparisons between vulvovaginal swabs and endocervical swabs using nucleic acid amplification tests for the detection of chlamydia 79 10 11 13 14 15 16 17 18 19 One included a comparison using two different nucleic acid amplification tests, making 12 comparisons in total 19. Two of the comparisons found a higher sensitivity with the endocervical swabs, with differences of 1.8%15 and 0.6%,11 whereas 10 found a higher sensitivity with the vulvovaginal swabs, with a median of 5.1% difference and a range of 1.2–16.0% 7…”

“…On transferring to the Aptima Combo-2 assay, we needed to choose between endocervical and vulvovaginal sampling. Most studies have shown that the two samples are equivalent,4 7 10 13 14 15 16 17 18 19 suggesting that, when a woman is undergoing an examination, an endocervical swab would be preferable on the assumption that it is superior for detecting low chlamydial load in the endocervix (which if missed could leave women at risk of ascending infection). However, one study found vulvovaginal swabs to be significantly more sensitive than endocervical swabs (81% v 65%), although the authors commented that the sensitivity of the endocervical swabs was lower than in previously published studies 9.…”

<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

“…38 Among the targets used to identify C. trachomatis, a cryptic plasmid has been the most widely used for NAATs. [39][40][41] However, it was subsequently shown that sequence variation of cryptic plasmids has been detected in several countries. [42][43][44] This demonstrates that false-negative results may occur when a cryptic plasmid is used alone as the target for detection of C. trachomatis particularly when screening clinical samples from this region.…”

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

“…There is no need for an initial denaturation step for DNA and the reaction has the ability to detect RNA [108][109][110][111]. HDA has been shown to be effective for various samples, from urine [112] and vaginal swabs [112] to blood [110,111] and stool [108,109]. Therefore, the HDA has a great potential for using it in portable pointof-care devices [113].…”