Gonorrhoea Nucleic Acid Amplification Tests Research Articles

Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility

To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid amplification test (NAAT)-positive clinical sample extracts (n = 40). The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical sample extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical samples confirmed to have WT gyrA sequences based on Sanger sequencing. These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.

Performance of human papillomavirus DNA detection in residual specimens taken for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid amplification testing in men who have sex with men

ObjectivesRectal swab specimens, either alone or pooled with first-void urine (FVU) and pharyngeal swab specimens, are used to test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection in men...

Epidemiology, clinical features and laboratory testing of Neisseria gonorrhoeae infections in the Australian capital territory in 2014–2018

Gonorrhoea is most commonly diagnosed by nucleic acid amplification tests (NAATs), however culture remains important for providing information on antimicrobial susceptibilities. This study describes the epidemiology of gonorrhoea in the Australian Capital Territory (ACT), clinician compliance with the performance of culture, and sensitivity of culture relative to NAAT. Positive Neisseria gonorrhoeae NAATs and culture and demographical information were identified from the ACT public pathology laboratory information system for a 5-year period (2014–2018). NAAT positive cases were reviewed to determine if a specimen had also been taken for culture. Repeat detection of N. gonorrhoeae from one or more sites within a 29-day period were regarded as a single episode. 917 episodes of N. gonorrhoeae involving 1,233 positive NAATs/cultures from urogenital (30%), throat (37%), anal (32%) and other sites (0.4%) were observed; 89% in males. An increase in number of episodes and test positivity rate were observed over time. 81% of NAAT positive specimens had culture performed, of which 64% isolated N. gonorrhoeae. Culture positivity was poorest for throat (57%), anal (65%), and vaginal (54%) specimens. Until other methods of detecting antimicrobial resistance are available, clinicians should be targeted to collect appropriate specimens for culture to inform treatment and surveillance.

Use of a Rapid Diagnostic for Chlamydia trachomatis and Neisseria gonorrhoeae for Women in the Emergency Department Can Improve Clinical Management: Report of a Randomized Clinical Trial

In emergency departments (EDs), diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae are challenging. We conducted a randomized clinical trial to assess rapid C trachomatis and N gonorrhoeae testing on overtreatment and undertreatment of women evaluated for C trachomatis and N gonorrhoeae. Women undergoing pelvic examinations and C trachomatis and N gonorrhoeae testing (n=254) were randomized to control or rapid test groups. The control group received standard-of-care C trachomatis and N gonorrhoeae nucleic acid amplification tests of endocervical specimens, with 2- to 3-day turnaround times. For the rapid test group, clinicians collected an extra endocervical swab for GeneXpert C trachomatis and N gonorrhoeae rapid testing, in addition to the standard-of-care nucleic acid amplification testswab. Rapid results were immediately provided, and all patients were treated according to providers' clinical judgment. In the rapid test group, 7.9% of patients had positive test results for C trachomatis; 3.9% had positive test results for N gonorrhoeae. In the control standard-of-care group, 10.2% of patients had positive nucleic acid amplification test results for C trachomatis; 5.5% had positive results for N gonorrhoeae. Undertreatment for both C trachomatis and N gonorrhoeae in the ED was 0% for the rapid test group and 43.8% for the control standard-of-care group. Clinicians overtreated 46.5% of uninfected standard-of-care control patients for C trachomatis compared with 23.1% of uninfected rapid test patients. For patients uninfected with N gonorrhoeae, clinicians overtreated 46.7% of standard-of-care control patients compared with 25.4% of rapid test patients. The length of stay did not differ significantly between groups. Rapid C trachomatis and N gonorrhoeae testing in the ED led to a significant reduction in overtreatment for women without infections compared with the standard-of-care control group. Additionally, in the rapid test group there was significant improvement in appropriate treatment for patients with infections.

Trends in chlamydia and gonorrhoea testing and positivity in Western Australian Aboriginal and non-Aboriginal women 2001-2013: a population-based cohort study.

This study aimed to examine trends in chlamydia and gonorrhoea testing and positivity in Aboriginal and non-Aboriginal women of reproductive age. A cohort of 318002 women, born between 1974 and 1995, residing in Western Australia (WA) was determined from birth registrations and the 2014 electoral roll. This cohort was then probabilistically linked to all records of chlamydia and gonorrhoea nucleic acid amplification tests conducted by two large WA pathology laboratories between 1 January 2001 and 31 December 2013. Trends in chlamydia and gonorrhoea testing and positivity were investigated over time and stratified by Aboriginality and age group. The proportion of women tested annually for chlamydia increased significantly between 2001 and 2013 from 24.5% to 36.6% in Aboriginal and 4.0% to 8.5% in non-Aboriginal women (both P-values <0.001). Concurrent testing was high (>80%) and so patterns of gonorrhoea testing were similar. Chlamydia and gonorrhoea positivity were substantially higher in Aboriginal compared with non-Aboriginal women; age-, region- and year-adjusted incidence rate ratios were 1.52 (95% confidence interval (CI) 1.50-1.69, P<0.001) and 11.80 (95% CI 10.77-12.91, P<0.001) respectively. Chlamydia positivity increased significantly in non-Aboriginal women aged 15-19 peaking in 2011 at 13.3% (95% CI 12.5-14.2%); trends were less consistent among 15-19-year-old Aboriginal women but positivity also peaked in 2011 at 18.5% (95% CI 16.9-20.2%). Gonorrhoea positivity was 9.7% (95% CI 9.3-10.1%), 6.7% (95% CI 6.4-7.0%), 4.7% (4.4-5.0%), and 3.1% (2.8-3.4%) among Aboriginal women aged respectively 15-19, 20-24, 25-29 and ≥30 years, compared with <1% in all age groups in non-Aboriginal women. Over time, gonorrhoea positivity declined in all age groups among Aboriginal and non-Aboriginal women. Between 2001 and 2013 in WA chlamydia and gonorrhoea positivity remained highest in young Aboriginal women despite chlamydia positivity increasing among young non-Aboriginal women. More effective prevention strategies, particularly for young Aboriginal women, are needed to address these disparities.

Gonorrhoea Diagnostic and Treatment Uncertainties: Risk Factors for Culture Negative Confirmation after Positive Nucleic Acid Amplification Tests.

Gonorrhoea incidence has increased substantially in Stockholm during the past years. These increases have coincided with changes in testing practice from solely culture-based to nucleic acid amplification tests (NAAT). Gonorrhoea NAAT is integrated with Chlamydia trachomatis testing and due to opportunistic screening for chlamydia, testing prevalence for gonorrhoea has increased substantially in the Stockholm population. The aim of this study was to examine epidemiological risk-factors for discordant case which are NAAT positive but culture negative. These discordant cases are especially problematic as they give rise to diagnostic and treatment uncertainties with risk for subsequent sequelae. All gonorrhoea cases from Stockholm county during 2011–2012 with at least one positive N. gonorrhoea NAAT test and follow-up cultures were included (N = 874). Data were analysed using multivariate and stratified logistic regression models. Results showed that women were 4-times more likely (OR 4.9; 95% CI 2.4–6.7) than men to have discordant cultures. Individuals tested for gonorrhoea without symptoms were 2.3 times more likely (95% CI 1.5–3.5) than those with symptoms to be discordant. NAAT method and having one week or more between NAAT and culture testing were also indicative of an increased likelihood for discordance. Using NAAT should be based on proper clinical or epidemiological indications and, when positive, followed-up with a culture-based test within one week if possible. Routine gonorrhoea testing is not recommended in low prevalence populations.

A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility.

The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

P13.11 Self-taken extragenital sampling for chlamydia and gonorrhoea in women – is it acceptable? feedback from a self-swab and clinician-swab trial

Introduction Self-taken vulvovaginal swabs analysed by nucleic acid amplification tests (NAATs) for chlamydia and gonorrhoea are standard practice worldwide. Extra-genital sampling (rectum and pharynx) is becoming increasingly important in women, with evidence that urogenital sampling alone may miss infections. Yet, little is known about women’s views of extra-genital sampling. We explored the acceptability of self-taken extra-genital samples for women as part of a clinician versus self-taken extra-genital sampling study. Methods Women attending a sexual health clinic were invited to participate in a ‘swab yourself’ trial. After randomisation, both clinician and self-taken samples for chlamydia and gonorrhoea NAATs from vulvovaginal, pharyngeal and rectal sites were taken. Participants were then invited to complete a questionnaire about the extra-genital sampling. Results 400 surveys were returned from 402 women recruited (response rate >99%), age range 17–57 (mean 25.2) years. 75% disagreed/strongly disagreed with the statement ‘taking my own samples was difficult’. 72% agreed/strongly agreed that they felt confident taking their own swabs but 30.5% agreed/strongly agreed they felt uncomfortable taking their own swabs; of these 53 (43%) stated they had never had anal sex. 42% agreed/strongly agreed that they would prefer to take their own samples compared to 34% who agreed/strongly agreed they would prefer clinician-taken swabs. 66% agreed/strongly agreed they would be happy to take the samples themselves in a non-clinic setting. Free comments covered themes of ‘more confidence if had clinician samples taken before’, ‘concerns if self-swabbing would give correct results’. Nine commented specifically on discomfort, but none disagreed with the statement ‘I would be happy to take my own swabs in a non-clinic environment’. Conclusion Extra-genital sampling was highly acceptable to the majority of women, with high levels of confidence and low reports of discomfort. This has positive implications for the future of extra-genital testing in women, especially in non-clinic settings. Disclosure of interest statement Dr Janet Wilson has received honoraria and travel and accommodation expenses from BD Diagnostics, and research grants in the form of diagnostic kits from Hologic/Gen-Probe.

Purification of DNA eluates from the ProbeTec GC Qx assay on BD Viper™ XTR allows for further analysis and confirmation of gonorrhea

Low positive predictive values of Neisseria gonorrhoeae nucleic acid amplification tests in low-prevalence populations are a major challenge for accurate diagnostics. It is therefore necessary to verify all positive N. gonorrhoeae results with a different assay and target gene, preferably using the same sample. The BD ProbeTec™ Q(x) Collection Kit for Endocervical or Lesion Specimens, which is recommended for BD Viper™ XTR, is incompatible with other commercial platforms. Therefore, a confirmatory PCR has not been available for samples received on this transport medium. To be able to verify results from these samples with another assay, our objective was to establish a procedure for using the DNA eluates from BD Viper™ XTR for further analysis. DNA eluates from BD Viper™ XTR were collected and analyzed in two in-house confirmatory real-time PCRs targeting the porA pseudogene and the opa multicopy gene. BD Viper™ XTR DNA eluates were analyzed directly and also after purification with the nucleic acid extraction system NucliSENS® easyMag®. Purification of BD Viper™ XTR DNA eluates with the nucleic acid extraction system NucliSENS® easyMag® provided a sensitivity of the in-house PCR comparable to BD Viper™. With the inclusion of two target genes in the confirmatory PCR, specific and reliable verification of results were obtained. This study presents a simple, inexpensive procedure which allows for rapid verification of gonorrhea from samples received on the BD ProbeTec™ Q(x) Collection Kit for Endocervical or Lesion Specimens.

P3.285 Diagnostic and Treatment Uncertainties: Epidemiological Risk Factors For NAAT Positive But Culture Negative Gonorrhoea Cases in Stockholm, Sweden

BackgroundThe advent of gene amplification testing methods for Neisseria gonorrhoea has lead to a higher prevalence of gonorrhoea testing in the population. Various methods for Nucleic Acid Amplification Tests (NAAT)...

P3-S1.08 Are there acceptable alternatives to syndromic management for the diagnosis of sexually transmitted infections in HIV positive Kenyan women?

BackgroundControl of sexually transmitting infections (STIs) is an important aspect of HIV prevention and care. Syndromic management for vaginal discharge, despite a recognised low sensitivity and specificity, is widely employed...

Nucleic Acid Amplification of the opa Gene for Detection of Neisseria gonorrhoeae: Experience from a Diagnostic Laboratory

We report results of Neisseria gonorrhoeae nucleic acid amplification testing (NAAT) with the Abbott m2000 PCR at a tertiary laboratory 6 months after its introduction. Of 5,475 specimens tested, 45 samples (0.82%) tested positive for N. gonorrhoeae. Eight were not cultured, but seven tested positive with a porA pseudogene NAAT.

Neisseria gonorrhoeae NAAT ? a problem down under

Nucleic acid amplification tests (NAATs) are used worldwide for the detection of Neisseria gonorrhoeae, either in conjunction with or in place of traditional bacterial culture techniques. There are numerous advantages of gonococcal NAATs, including increased sensitivity, that a viable organism is not needed for detection, and they can be used effectively on non-invasive specimens such as urine and self-collected specimens. For these reasons, NAATs have been particularly useful for patients in remote regions of Australia where sexual health services may not be available and where religious or cultural restrictions otherwise restrict opportunities for specimen collection. Australian studies have been at the forefront of investigating the use of self-collected NAAT specimens and particularly successful at introducing the use of tampon self-collected specimens in remote populations of Indigenous Australians.

A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection.