Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) User Manual K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Purpose The Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) is commissioned by Health Protection Scotland (HPS) to provide a national reference service for the management of bacterial sexually transmitted infections throughout Scotland. Scope of service The following services are provided for referred isolates of presumptive Neisseria gonorrhoeae: • Identification of ‘problem’ isolates • Antibiotic resistance surveillance • Molecular typing of gonococci by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) and, where appropriate, by other methods • Characterisation of medico-legal isolates • Identification of N. gonorrhoeae by nucleic acid amplification test (NAAT) for non-viable isolates. The following tests are performed on clinical specimens: • Supplemental NAAT for N. gonorrhoeae (GC NAAT) • Real-time PCR for T. pallidum on exudates from ulcers that are suspected to be syphilitic • Real-time PCR for Lymphogranuloma venereum (LGV) on Chlamydiapositive samples from cases of suspected LGV. The SBSTIRL also provides these general services: • Clinical and microbiological advice • Assessment of new methodologies • Participation in national/international surveillance and quality assurance projects • Provision of six-monthly reports to users • Education, training, audit and collaborative research K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Contact details Name and Designation Director Dr Kirstine Eastick Deputy Director and Medical Microbiology Adviser Dr A Paddy Gibb Telephone numbers and Email address 0131 242 6032 [email protected] [email protected] 0131 242 6070 [email protected] Specialist Biomedical Scientists Mr John Anderson Mr Brian Malloy Ms Alison Fleming 0131 242 6078 Administrator Mrs Janis Shade 0131 242 6081 [email protected] Departmental confidential fax 0131 242 6008 The service is managed overall by NHS Lothian. If we are unable to deal with an issue and you wish to discuss a matter with NHS Lothian Management then contact: Mr Mike Gray Tel: 0131 242 6076 [email protected] Laboratory Service Manager, NHS Lothian Royal Mail Postal Address Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) Microbiology Department Edinburgh Royal Infirmary 51 Little France Crescent Edinburgh EH16 4SA Hays DX Address DX 6231202 Edinburgh 96EH K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Hours of work Core laboratory hours are 9am – 5pm Monday to Friday. There is no service on Public Holidays or weekends. Submission of samples Please complete the relevant SBSTIRL request form (see end of document). Printed request forms can be provided by SBSTIRL upon request. There are four separate request forms for submission of N. gonorrhoeae cultures, specimens for supplemental N. gonorrhoeae NAAT, T. pallidum and LGV real time PCRs. All specimens must be labelled with the following information: Unique Identifier (Surname & Forname/CHI number/NaSH number) Date of birth Sender’s Laboratory Number All specimens must be accompanied by the appropriate request form with the following information: Patient’s unique Identifier (Surname & Forname/CHI number/NaSH number) Patient’s date of birth and gender Specimen type/site Sender’s Laboratory Number Consultant to whom a report should be addressed Location of Sender Any relevant clinical information The specimen types that are appropriate for each reference test are outlined in the table below K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Table of Specimen types appropriate for each reference test Test N. gonorrhoeae culture, identification, sensitivities and typing Specimen type ‘Chocolate agar’ slope of the culture after a minimum of 6 hours incubation at 35-37ºC, 5% CO2. or Overnight growth of organism transferred to an Amies charcoal agar transport swab Supplemental GC Any positive or equivocal GC NAAT specimen NAAT in any media as recommended by the NAAT manufacturer N. gonorrhoeae Any NAAT specimen which is GC positive by typing directly from a two different NAATs or two different targets clinical specimen within the same NAAT T. pallidum PCR Any swab from ano-genital or oral ulcers/lesions that are suspected to be syphilitic, in a liquid transport medium or dry. Lymphogranuloma venereum (LGV) PCR Any NAAT specimen that is chlamydia positive K Eastick March 2012 Review date: March 2014 Comment Please ensure this arrives Monday-Friday. Viability in transit cannot be guaranteed beyond 2 days. Please note that there is no delivery service on Public Holidays. Please indicate on supplemental GC NAAT request form which test has already been performed and the result. A supplemental GC NAAT is not required but the specimen is very useful for sequence typing (and prediction of antibiotic susceptibility) if no cultured isolate is available. Use the supplemental GC NAAT request form. Use a T. pallidum PCR request form. Tests are charged to the requesting laboratory at £25 per specimen. Use an LGV PCR request form. Suitable specimens are those from symptomatic patients, contacts of LGV and chlamydia-positive rectal swabs from HIV-positive patients. PD-MIC-R-STIUSR Ver 2.0 Tests performed Test Methodology Frequency Turnaround time N.gonorrhoeae culture identification Oxidase test Gram stain Rapid Carbohydrate Utilisation Test (including βlactamase test); API NH test when necessary Nitrocefin test Phadebact Minimum inhibitory concentration for 7 antibiotics Daily 4 days Twice weekly 7 days Weekly 11 days Daily 3 days Monthly 12 weeks Weekly Weekly 7 days 7 days N. gonorrhoeae antibiotic susceptibility Typing NG-MAST (Neisseria gonorrhoeae multi-antigen sequence typing) on all episodes of infection Opa typing (when required) Supplemental GC Assays targeting PorA (in-house realtime PCR) and 16s NAAT RNA (Aptima GC, Genprobe) N. gonorrhoeae NG-MAST using nested PCR to achieve sensitivity typing directly on clinical specimens T. pallidum PCR Real time PCR detecting PolA gene Real time PCR detecting pmpH gene LGV PCR Turnaround time excludes weekends and public holidays Further description of these methodologies is given below: K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Methods used at SBSTIRL N. gonorrhoeae 1. Identification The identity of all referred cultures of N. gonorrhoeae is confirmed using the following tests: Oxidase test: Positively identifies organisms that constitutively produce cytochrome oxidase Gram stain: Gram negative diplococci are identified for further testing Rapid Carbohydrate Utilisation Test (RCUT): Utilisation of glucose and not maltose, sucrose, lactose or fructose distinguishes N. gonorrhoeae from other oxidase-positive Gram-negative diplococci. A test for β-lactamase is included in this assay, which identifies penicillinase producing Neisseria gonorrhoeae (PPNG). Phadebact GC monoclonal test: A species-specific co-agglutination reaction between monoclonal antibody reagents with the major porin protein positively identifies N. gonorrhoeae and subdivides strains into two serogroups WI or WII/III types. API NH test: A panel of biochemical tests; this is performed only if RCUT or Phadebact results are inconclusive. 2. Antibiotic sensitivity All positively identified N. gonorrhoeae isolates are tested by the agar plate dilution method for their sensitivity to the following seven antibiotics: Azithromycin (0.015-2mg/L) Penicillin (0.015-2mg/L) Cefixime (0.015-0.12mg/L) Spectinomycin (16-128mg/L) Ceftriaxone (0.015- 0.12mg/L) Tetracycline (0.25-4mg/L) Ciprofloxacin (0.004-1mg/L) E-tests are used for further analysis of isolates with MICs that exceed the concentration range covered by the agar plate dilution and for those that give an MIC at a resistance category threshold. 3. Sequence typing Molecular typing is performed using the Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) method (Martin et al. JID 2004:189;1497-1505). Sequence typing is performed on all episodes of gonorrhoea. A patient with multiple sites of infection has typing performed on genital, oral and rectal specimens. NG-MAST is also performed on clinical specimens that are N. gonorrhoeae positive by NAAT if no cultured isolate from the patient is available (where sufficient DNA is present). NG-MAST is a highly discriminatory typing scheme that differentiates between isolates on the basis of the sequence variation observed in two genes coding for the por protein and transferrin binding protein b. Each sequence type (ST) is unique and differs from any other by at least one nucleotide. Clusters of isolates sharing the same ST can be observed over time and both phenotypic K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 and demographic data support the notion that the clusters are valid, containing related isolates. 4. Supplemental NAAT testing for N. gonorrhoeae A second test that targets a different specific gene is required to ensure a true positive GC result. We provide alternative assays to ensure that what ever the first GC NAAT used at your laboratory, there will be a suitable second test available for supplemental testing. Assays currently in use are an an in-house PCR which targets the PorA pseudogene and the Aptima GC test (GenProbe), which targets N. gonorrhoeae 16s RNA. 5. Additional tests NAAT for non-viable cultures Material from any referred isolate that fails to grow is tested for the presence of N. gonorrhoeae DNA. Currently the Gen-probe Aptima GC test is used. Opa typing (O’Rourke et al Mol Microbiol 1995:17;865-75). This is a restriction fragment length polymorphism (RFLP) method that may be used to supplement the discrimination achieved by NG-MAST typing on selected groups of isolates where necessary. Tests for other Sexually Transmitted bacterial infections T. pallidum PCR A real-time PCR to detect a region of the DNA polymerase A gene is performed (Koek et al Clin Micro Infect 2006 12:1233-6) and uses an ABI 7500 platform. This test is not funded under our Service Level Agreement with Health Protection Scotland, and requesters will be charged £25 per specimen tested. LGV PCR The presence of C. trachomatis DNA is first confirmed by a nucleic acid amplification test. A real-time PCR to detect the polymorphic membrane protein H gene is performed (Morré et al. Emerge Infect Dis 2005:11;1311-2.) and uses an ABI 7500 platform. Referred tests Occasionally, specimens and isolates may be referred to the Sexually Transmitted Bacteria Reference Laboratory at the Health Protection Agency’s Centre for Infections, Colindale, for further specialist testing unavailable in Scotland. All such referrals should be made through SBSTIRL in order to be funded by HPS. K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Additional tests Gonococcal isolates and medicolegal specimens are stored indefinitely, and DNA extracts from specimens for NAAT are stored for at least one year. For retrospective testing or typing within these time-frames, please contact the laboratory. Reporting of Results Written reports are issued and sent to the requesting consultant via mail. For gonococcal cultures, a preliminary report of identification is issued followed by a further report of antimicrobial susceptibility and sequence type. NAAT reports are issued as soon as possible. For NAAT-positive samples an updated report containing sequence typing data will be issued at a later date where there are no culture isolates for an episode of infection. Positive T. pallidum PCR and LGV PCR results are telephoned directly to the requesting consultants. The laboratory is currently introducing a facility for the transmission of results by email. Accreditation details SBSTIRL, as part of the Department of Laboratory Medicine, Microbiology Laboratory, has been accredited by CPA (CPA Reference Number 2496) and found to be in compliance with “Standards for the Medical Laboratory” incorporating ISO 15189:2003. External audit schemes National External Quality Assurance (NEQAS) SBSTIRL, as part of the Department of Laboratory Medicine, Microbiology Laboratory participates in NEQAS. Quality Control for Molecular Diagnostics (QCMD). SBSTIRL participates in the QCMD scheme for N. gonorrhoeae NAATs, which produces one distribution of 10 samples annually. Quality assurance exchange with the Health Protection Agency (HPA) SBSTIRL exchanges the following specimens with the Sexually Transmitted Bacteria Reference Laboratory (STBRL) at the HPA: Specimen 20 gonococcal isolates Test Antimicrobial susceptibility 5 gonococcal DNA lysates 10 ulcer specimens 10 swab specimens NG-MAST T.pallidum PCR LGV PCR K Eastick March 2012 Review date: March 2014 Frequency Twice each year Annually Annually Annually PD-MIC-R-STIUSR Ver 2.0 SBSTIRL activities and dissemination of data The SBSTIRL works closely with colleagues in microbiological clinical and epidemiological disciplines to provide timely and relevant data; where possible this is both collected and disseminated in electronic formats. Staff members make every effort to attend national and international meetings within the bacterial STI field to present work and maintain awareness of the most up to date findings. The reference laboratory activities are summarized in the organogram below: Gonococcal Antibiotic Surveillance in Scotland Data published in Participation in HPS Weekly Report national & annual report (GASS) Six-monthly reports to users by electronic format international STI working groups Collection of Antimicrobial Resistance testing of all isolates Quarterly data epidemiological data submission to HPS (via STISS) Quality assurance by established EQA or exchange programmes Dissemination of Data Scottish Bacterial STIs Reference Laboratory Audit of gonococcal Presentations at conferences & peer reviewed publications antibiotic resistance levels in Scotland Sequence typing of all cases of gonorrhoea Consultation with BASHH Scottish branch Recommendation for change Supplemental NAAT testing for gonorrhoea Real time PCR for T. pallidum from ulcer material. Real time PCR for LGV in treatment guidelines Surveillance report of Identification of problem isolates K Eastick March 2012 Review date: March 2014 Weekly summary of typing data sent directly to GUM clinics positive cases to HPS PD-MIC-R-STIUSR Ver 2.0 Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) Telephone 0131 242 6032 Fax:0131 242 6008 Hays DX: DX 6231202, Edinburgh 96EH To: From: SBSTIRL Microbiology Department Edinburgh Royal Infirmary 51 Little France Crescent Edinburgh EH16 4SA REQUEST Date Taken: Date Posted: Case No/Patient: CHI No: Category of patient: DOB: / Sex: M Specimen/ Site GUM GP Hospital / F Beta-Lactamase producer Sender’s Lab Reference No For SBSTIRL use Urethra Cervix Rectum Throat Others (Please state) _______________________________________________________________ Comment: Please supply details of clinician whom we can contact for epidemiological information Name: Address: K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Supplemental Neisseria gonorrhoeae NAAT Request Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) Microbiology Department, Edinburgh Royal Infirmary 51 Little France Crescent, Edinburgh EH16 4SA Hays DX: DX 6231202, Edinburgh 96EH FROM: Microbiology Laboratory Consultant Name Address Tel: ______________________________________________________________ REQUEST Date taken: Date posted: Case No/Patient: Category of patient: GUM DOB GP Hospital Sex: M F ______________________________________________________________ SPECIMEN: The referred specimen should be N. gonorrhoeae positive or equivocal by your own NAAT. Please send : a) your sample transferred to an aptima urine or swab collection tube or b) the remainder of the clinical specimen Your Laboratory Number:……………………………………………………………. Specimen type The GC NAAT you used Abbott real time CT/NG Aptima Combo 2 BD Probetec Roche Cobas 4800 Other (specify target) Cervical Vulvo-vaginal Urine Urethral Rectal Throat Other (specify) Your GC NAAT result Positive Other (specify) Positive Other (specify) Positive Other (specify) Positive Other (specify) Positive Other (specify) Was the NAAT result reproducible? Yes No Not done Sequence typing only required as GC NAAT already confirmed ? ___________________________________________________________________ For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab) K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Treponema pallidum PCR Test Request Scottish Bacterial Sexually Transmitted Infections Reference Laboratory Microbiology Department, Edinburgh Royal Infirmary 51 Little France Crescent, Edinburgh EH16 4SA Hays DX: DX 6231202, Edinburgh 96EH FROM: Microbiology Laboratory Consultant Name Referring Clinic (e.g. GUM) Consultant Name Address Address Tel: Tel: ______________________________________________________________ REQUEST Date taken: Date posted: Case No/Patient: DOB: Sex M F ______________________________________________________________ SPECIMEN: A swab of exudate from the depth of the lesion in virus transport medium Site of suspect primary lesion: Genital Anal Oral Other (specify) __________________________________________________________________________ ADDITIONAL INFORMATION: Known contact of patient with syphilis Yes No Signs/Symptoms Yes No If yes, please give details………………………………………………………………. Epidemiological treatment Yes No If yes, please give treatment and date….……………………………………………….. ………………………………………………………………………………………….. Dark ground microscopy Positive Negative Not done For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab) K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0 Lymphogranuloma Venereum (LGV) Test Request Scottish Bacterial Sexually Transmitted Infections Reference Laboratory Microbiology Department, Edinburgh Royal Infirmary 51 Little France Crescent, Edinburgh EH16 4SA Hays DX: DX 6231202, Edinburgh 96EH FROM: Microbiology Laboratory Consultant Name Referring Clinic (e.g. GUM) Consultant Name Address Address Tel: Tel: ______________________________________________________________ REQUEST Date taken: Date posted: Case No/Patient: DOB Sex: M F ______________________________________________________________ SPECIMEN: The referred specimen should be chlamydia positive Your Laboratory Number: Specimen site (specifiy) Chlamydia test used SDA Urethral Rectal Taqman/Cobas Aptima Combo Abbott real time CT/NG Other Other (specify) __________________________________________________________________________ REASON FOR REQUEST: Symptomatic patient Proctitis Inguinal Lymphadenopathy Other (specify) LGV contact Chlamydia positive rectal swab from HIV positive patient For further information please contact 0131 242 6032 (Director), 6081 (Secretary), or 6078 (Lab) K Eastick March 2012 Review date: March 2014 PD-MIC-R-STIUSR Ver 2.0