Guidance on the use of molecular testing for Neisseria gonorrhoeae in Diagnostic Laboratories 2011
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1 Guidance on the use of molecular testing for Neisseria gonorrhoeae in Diagnostic Laboratories 2011 Molecular testing for gonorrhoea working group (see Appendix) Prepared by Kirstine Eastick March
2 Summary Nucleic acid amplification tests (NAATs) for Neisseria gonorrhoeae (GC) are the most sensitive methods for diagnosis, and should be the Standard of Care in Scotland. No commercially available GC NAAT is CE marked for use with extragenital specimens. However, due to the increased sensitivity over culture at these sites, laboratories should validate and use NAATs for the diagnosis of extragenital infection. All reactive GC NAAT tests should be repeated using a second test, with a different gene target to the screening test. This may be performed at the Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) or locally. If, after analysis of local data, the positive predictive value of the primary test is >90%, (calculated by anatomical site and setting), then a preliminary result may be issued to the requester by the testing laboratory. Cultures, by direct plating if possible, should be taken from patients who are: symptomatic, positive on gram film, contacts of gonorrhoea or suspected of failing therapy, and any other patients positive by NAAT. Positive specimens by NAAT, and cultures of N. gonorrhoeae, should be sent to SBSTIRL. NHS Boards should have robust procedures in place to ensure that specialist Sexual Health services are aware of all individuals diagnosed with gonorrhoea 1. Background information Introduction Nucleic acid amplification techniques (NAATs) have been the test of choice for the diagnosis of Chlamydia trachomatis in Microbiology laboratories for around ten years. A number of commercially available platforms now provide combined testing for both C. trachomatis and N. gonorrhoeae in a single test. Some laboratories can now offer in-house NAATs for these organisms. Molecular techniques allow the testing of less invasive sample types, have less stringent transport requirements than culture, and are more sensitive. They are now accepted as the Standard of Care for gonorrhoea testing in Scotland. Due to increased sensitivity, use of these methods will inevitably lead to gonorrhoea diagnoses where there is no N. gonorrhoeae isolate. However, continuation of a culture service in Sexual Health clinics and Microbiology laboratories is essential in order to provide antimicrobial susceptibility data to inform gonorrhoea treatment strategy into the future. This document is an update on Guidance on the introduction of molecular testing for Neisseria gonorrhoeae in diagnostic laboratories produced jointly by HPS and the SBSTIRL reference laboratory in This update reflects the current position, whereby combined testing is now available in the majority of NHS Boards, and extends guidance beyond the introduction of these tests to include recommendations for testing of extragenital samples, for the issuing of preliminary reports and for referral of positive patients to Sexual Health services. 1 Improving sexual health services in Scotland: Integration and Innovation. National Overview Healthcare Improvement Scotland
3 Nucleic Acid Amplification tests (NAATs): The sensitivity and specificity of these tests varies between manufacturers but all have a proportion of false positive results. In populations with a low incidence of the infection this proportion becomes significant and therefore needs to be taken into account. There is also a risk that, due to the variable nature of Neisseria genomes, and the likelihood of genetic transfer between them, false-positive results will be produced when commensal Neisseria species are present 2. These bacteria are most often found colonising the throat and, less frequently, the rectum. The optimal method for confirming a positive NAAT result is to perform a second supplemental NAAT test on a different platform using a test that detects a different target. Confirmatory testing is available at SBSTIRL, but some laboratories may prefer to perform this in-house. Due to the extremely high sensitivity of NAATs, care should be taken both in the laboratory and the clinic to prevent cross-contamination of samples. It is possible to sequence strains of N. gonorrhoeae from NAAT-positive samples. Typing of strains is important for contact tracing strategies and national surveillance. It is not currently possible to test samples directly for resistance determinates due to the genetic complexity of emerging cephalosporin resistance and the possibility that resistance genes are carried by bacteria other than N. gonorrhoeae in the sample. The probability of a resistance profile may be estimated, based on the sequence type of a strain without culture. This does not allow identification of new phenotypic resistance patterns. The continuation of culture-based surveillance is therefore vital. N. gonorrhoeae culture: Culture for N. gonorrhoeae is technically and logistically demanding, and yields fewer gonorrhoea diagnoses than GC NAAT, particularly at extragenital sites. However, it is essential that isolates are obtained from as many episodes of gonorrhoea as possible, in order to allow antibiotic resistance surveillance. Isolation rates of 70% for genital samples, and 40% for rectal samples should be achievable. Although the yield from pharyngeal swabs is likely to be low, culture should be attempted as treatment failure has been more frequently reported in pharyngeal infection. An archive of Scottish isolates is maintained at SBSTIRL providing a valuable resource for retrospective studies which would not be possible with clinical specimens. Resistance surveillance is an essential part of the national surveillance programme for N. gonorrhoeae. Changes in resistance profiles guide changes in first line management of cases. In Sexual Health clinics, patients presenting with a high probability of gonorrhoea infection are treated prior to a full antibiotic sensitivity result; such epidemiological treatment is based on guidance produced as a consequence of national antimicrobial resistance studies. 2 Tabrizi et al., Evaluation of six nucleic acid amplification tests for detection of Neisseria gonorrhoeae and other Neisseria species. Journal of Clinical Microbiology 49(10):
4 Once resistance to an antibiotic exceeds 5% of isolates, first line treatment needs to be changed. To better understand the extent of resistance it is helpful to stratify cases according to the origin of their infection e.g. acquired locally or overseas. A high number of resistant strains in one area may skew the overall resistance data within Scotland leading to an inappropriate change in recommendations for first line prescribing. Given the sample size for Scotland (approximately episodes per annum), a significant drop off in numbers could alter the resistance profiles in subsets such as individual NHS Boards or routes of transmission. Sampling by anatomical site: Until the introduction of dual testing with GC, most chlamydia NAAT testing for both men and women was performed using urine samples. In women, depending on the particular test, up to 25% of cases of gonorrhoea can be missed using urine rather than a cervical swab 3. As self-taken vulvovaginal swabs perform as well as or better than clinician-taken endocervical swabs in detecting gonococcal and chlamydial infection either of these specimens is acceptable. Urine remains the specimen of choice in men for NAAT testing. More detail on suitable specimen types can be found in UK and European guidance (Appendix 2), as well as in manufacturers recommendations for individual kits. None of the commercially-available GC NAAT tests are CE marked for use with extragenital samples, which may include throat/pharyngeal swabs, rectal swabs, eye swabs and other samples from suspected disseminated gonorrhoea. The increased sensitivity of NAATs for the detection of pharyngeal and rectal infection is particularly valuable and is recommended (see UK and European guidance, appendix 2). However, a local validation must be carried out in the laboratory prior to the introduction of testing. Confirmatory testing of positive extragenital samples with a second GC NAAT targeting an alternative gene is mandatory prior to release of the final test report. Dual testing and positive predictive values The positive predictive value (PPV) (that is, the percentage likelihood that a positive result is a true positive) of the test is influenced by the specificity of the test (which may vary by specimen type, as discussed above), and the prevalence of the infection in the population. The prevalence of chlamydia and gonorrhoea are very different and the different components of a dual test will therefore also have markedly different PPVs 4. 3 Gaydos et al., Perfomance of the Abbott RealTime CT/NG for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Journal of Clinical Microbiology 48(9): Guidance for gonorrhoea testing in England and Wales. (2010). BASHH & HPA
5 The availability of combined tests means that there is now in effect a STI screen available for use in the primary care setting. The PPV for GC NAAT in this setting is likely to be lower than that observed in the specialist sexual health setting and therefore the PPVs for the two populations should be calculated separately by each NHS board for each testing laboratory. Laboratories need to be clear about how they will interpret tests for N. gonorrhoeae in this context given the potential for, and implications of, a false positive result in a population with a low incidence and unsuspecting of the finding. It is recommended that laboratories await confirmation of a positive NAAT result before issuing a report if the PPV is <90%. For populations and specimen types where the locally calculated PPV of the screening test is >90%, an interim report may, following local agreement, be issued to the requesting health care staff. This allows for a shorter turnaround time. SBSTIRL will issue regular data on confirmation rates to NHS Boards to facilitate these calculations. The PPV for a given test, specimen type and population is likely to be dynamic. Transient increases in prevalence, e.g. during an outbreak in the community, may influence the PPV, and this may trigger the issue of interim reports while the prevalence of infection is raised. In this situation it is envisaged that shorter turnaround times would aid in the management and control of such an outbreak. The reference laboratory should be informed of increases in prevalence and can advise on the likely effect on PPV
6 Recommended protocol for use of GC NAATs 1. Choice of method Method may be determined by the platform already available in the Laboratory, and the choice of method may be limited by contractual obligations. The SBSTIRL provides advice on the quality of commercial tests available and pointers for consideration when implementing a new test. 2. Introduction of testing Introduction of GC NAATs should be done in discussion with a local genitourinary medicine consultant for services and the Lead Clinician for Sexual and Reproductive Health. During introduction of the test the SBSTIRL will provide advice and confirmation of results. It is especially important to validate the test locally for use with extragenital samples. SBSTIRL can provide advice and help during this process, and may be able to facilitate the sharing of data between laboratories using the same test. 3. Reporting results (see also Flow Chart) The specificity of NAATs for gonorrhoea is not absolute, some tests are prone to crossreaction with other Neisseria species; a positive test should be confirmed using a second assay, targeting an alternative gene. Due to the high test sensitivity a negative test can reliably exclude gonorrhoea when an appropriate sample is used. Local agreement should be reached with sexual health services on reporting of preliminary results if the locally-calculated PPV of the test exceeds 90% for the specimen type in question. It is unlikely that the PPV of the test will exceed 90% outwith the specialist sexual health setting due to the low prevalence. Confirmation of a positive result should be awaited before reporting in this situation. Turnaround time (from receipt in the laboratory to release of report) for specimens should be 2 working days for negative results 5 working days for confirmed positive results The overall turnaround time should be no more than seven working days (BASHH/HPA guidance, Appendix 2)
7 All positive samples should be forwarded to SBSTIRL, even if confirmed locally, for sequence typing. Patients diagnosed outwith specialist settings should be referred to sexual health services to ensure adequate treatment and partner notification. NHS Boards should have a system in place to ensure that specialist services are aware of every patient diagnosed with gonorrhoea, and that the referral rate is auditable against Healthcare Improvement Scotland Standards (Appendix 2). Sexual health services should be involved in the management of patients whose specimens are unconfirmed reactive, as repeat testing, careful history-taking to assess risk and sensitive handling of partner notification are essential. (QIS Standard 4, Appendix 2). Clinicians should regularly review these cases, in order to inform clinical judgement. SBSTIRL should be informed of unusual occurrences which may point to mutations causing shifts in the specificity or sensitivity of the NAATs used. Role of culture A policy of selective culture is cost-effective as it leads to a large reduction in culture workload while still obtaining isolates from a majority of episodes. Patients attending sexual health clinics should have swabs taken for culture, in addition to NAAT screening, in the following circumstances: - Any patient symptomatic for gonorrhoea Any patient with gram negative diplococci in the gram film Any cases of gonococcal treatment failure Any contact of gonorrhoea All return patients who are NAAT positive not covered above o Culture from NAAT-positive sites if untreated Particular effort should be made to obtain a culture from those in the above categories who are likely to have acquired their infection abroad. Test of Cure (ToC) may be performed by culture or NAAT, but in the event of a NAATpositive ToC specimen, every effort should be taken to obtain an isolate for susceptibility testing. Each NHS board should aim to isolate GC from 70% of genital and 40% of rectal infections. Isolation rates from pharyngeal infections are likely to be low, but culture should be attempted as gonorrhoea at this site is less likely to respond to treatment
8 Culture-positive, NAAT negative patients (at corresponding anatomical sites) may indicate a problem with the NAAT test, and should be audited regularly. All isolates should be sent to SBSTIRL for antibiotic sensitivity testing and sequencing. Role of the Reference Laboratory SBSTIRL will: Advise on commercial systems available, their strengths and drawbacks Support laboratories during test validation, and encourage Scottish laboratories to share validation data. Provide supplemental testing during the period of introduction of NAATs until an acceptable level of concordance between laboratory results is established. Provide ongoing supplemental testing for labs that detect only one target (see implications for turnaround times above). Turnaround 95% of NAAT confirmations within 3 working days of receipt. Work with laboratories to provide reports in electronic format to reduce turnaround times. Sequence type all strains either from NAAT or culture to support local contact tracing and national antimicrobial resistance surveillance. Perform sensitivity testing on all isolates received to advise on individual treatment and to support the national antimicrobial resistance surveillance programme. Annually calculate the NAAT confirmation rate for each testing laboratory and communicate to NHS Boards, along with genital and extragenital culture rates - 8 -
9 NAAT testing flowchart recommended for all laboratories (1) Test sample by NAAT Unreactive /negative Indeterminate /equivocal Reactive Report: Neisseria gonorrhoeae nucleic acid not detected. Repeat according to manufacturer s instructions Indeterminate /equivocal Unconfirmed/ indeterminate Report: The result is unclear and the test should be repeated unless the patient has already been treated. Send to SBSTIRL for confirmatory testing* Reactive Optional process: Retest using a different target gene Optional process: If PPV for this test, sample type, and population >90%, consider issuing a preliminary report. Refer patient to sexual health service * See Flowchart (2) Report: Neisseria gonorrhoeae nucleic acid detected. Refer patient to sexual health service. Culture of affected site recommended before treatment. Inform NHS Board. Perform NG-MAST at SBSTIRL if no isolate for this episode
10 NAAT testing flowchart (2) SBSTIRL confirmatory algorithm Test sample using inhouse PCR a in duplicate Reactive x2 Other result Report: N. gonorrhoeae nucleic acid present. This result confirms the original NAAT test. Perform NG-MAST on one sample from each episode without a typed isolate Positive in second-line assay OR 2 replicates of in-house assay c PCR c Retest using in-house PCR (reextract if volume allows) And Test on second line assay b (if volume allows) Report: Indeterminate result, please repeat if clinically relevant. d a First-line assay 5 currently amplifies sequences from the PorA pseudogene of N. gonorrhoeae and includes an internal inhibition control. b Second-line assay is currently GenProbe Aptima GC, which amplifies rrna from N. gonorrhoeae and does not control for inhibition. c Can be either 2 of 2 replicates on repeat, or1 of 2 replicates from each of two separate extracts d Other comments may be added if the sample was inhibitory or insufficient for the second-line test Second-line test equivocal and/or 1 of 2 replicates positive on inhouse PCR All tests negative Report: This result does not confirm the original NAAT test. Consider repeat if clinically indicated. d 5 Whiley & Sloots 2005 Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies. Pathology 37:
11 Appendix 1 Membership of the working group Dr Kirstine Eastick, Director, SBSTIRL (chair) Dr Andrew Winter, Consultant in Genitourinary Medicine & HIV, NHS Greater Glasgow and Clyde, BASHH Dr Lesley Wallace, Epidemiologist, Health Protection Scotland Dr Kirsty Abu-Rajab, Consultant in Genitourinary Medicine & HIV, NHS Forth Valley Dr Dave Hamilton, Consultant Microbiologist, NHS Dumfries and Galloway Dr Dave Yirrell, Consultant Clinical Scientist in Virology, NHS Tayside Dr A Paddy Gibb, Consultant Microbiologist, NHS Lothian and Deputy Director, SBSTIRL Appendix 2 Other Relevant Guidance Detection of Neisseria gonorrhoeae using molecular methods. (2010). QSOP 62, Health Protection Agency. Guidance for gonorrhoea testing in England and Wales. (2010). BASHH & HPA. Sexual health services, standards. (2008). Quality Improvement Scotland (now Healthcare Improvement Scotland) da10f731f19&version=-1 UK National guideline for the management of gonorrhoea in adults (2011). Clinical Effectiveness Group, BASHH European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults. (2009). C Bignell. International Journal of STD and AIDS 20:
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