Chlamydia NAATs: update in the clinical and laboratory setting Gill Underhill St Mary’s Hospital Portsmouth

Chlamydia trachomatis – – – – Obligate intracellular gram negative organism Two forms: Infectious EB – elementary body – spore like Replicating RB – reticulate body – metabolically active From

Chlamydia trachomatis Commonest bacterial STD Spread by direct contact Incubation period 7 – 14 days Disease due to direct damage to cells and immunopathology causing fibrosis and scarring Infection does not protect against re-infection

Chlamydia Infection in Men Asymptomatic infection ~ 50% Non specific urethritis Strong associations with: Acute epididymitis Prostatitis Male infertility

Chlamydia Infection in Women Asymptomatic infection ~ 70 % Mucopurulent cervicitis Urethral infection Pelvic inflammatory disease in up to 40% - ascending infection involving uterus, fallopian tubes, and other pelvic structures Complications include chronic pelvic pain, ectopic pregnancy and < 20% have infertility Fitz-Hugh-Curtis syndrome – peri-hepatitis Peri-splenitis, peri-nephritis, peri-appendicitis

Other Diseases Proctitis – especially homosexual men Reactive arthritis – acute onset. Involves mostly knees, ankles, toes Reiter’s syndrome – mainly men - iritis, uveitis, conjunctivitis, and urethritis Neonates infected during birth ~ 20% conjunctivitis and/or pneumonia

Treatment of Chlamydia Tetracycline for 7 days Erythromycin for 7 days in pregnancy Azithromycin – single dose for patient compliance problems but more expensive Neonates – erythromycin for 10 days

Diagnostic Options for Chlamydial Infections Cell Culture Direct Fluorescence Assay Enzyme Immunoassay Hybridisation Assay Nucleic Acid Amplification Tests

Cell Culture Original gold standard High specificity ~100%, sensitivity 65-80%. Continuous cell lines - McCoy Incubate hours - slow turnaround Stain intracytoplasmic inclusions with fluorescein labelled monoclonal antibody Labour intensive and expensive Viable organisms – transport /storage critical Not suitable for large workload – manual method

Direct Immunofluorescence Smear of swab stained with fluorescein conjugated specific monoclonal antibody Rapid turnaround (2 hours) Specificity high ~ 95% Sensitivity 70 – 80% Relatively inexpensive Viable organisms not required Not suitable for large workloads – manual method

Enzyme Immunoassay Standard antigen capture EIA Sensitivity 60 – 80% Lower specificity ~ 90% - false positives Blocking assay improves specificity Fast turnaround time 3 – 5 hours Suitable for large workload and inexpensive Viable organisms not required Most popular test until recently

Nucleic Acid Amplification Tests Amplify nucleic acid sequence specific for organism High Sensitivity ~ 95% – can detect single copy DNA/RNA High specificity 99 – 100% Viable organisms not required Rapid turnaround time (3 – 5 hours) Suitable for urines

Nucleic Acid Amplification Tests Technically demanding Special areas required Contamination problems Specimen transport and storage critical Expensive No confirmatory test PCR, SDA, TMA, (LCR)

Polymerase Chain Reaction Roche Amplicor first commercially available PCR. Target is chlamydia plasmid. Primers are biotin labelled to produce biotin labelled amplicons

Cobas Amplicor Denaturation & Hybridization Denaturation Denatured Amplicon Strands Biotin Magnetic Microparticles Capture Probe D-cup A-Tube Wash Wheel

Cobas Amplicor Conjugate & Substrate Addition Avidin-horseradish Peroxidase Conjugate forming complex with Biotin Tetramethylbenzidine (TMB) Substrate Hydrogen Peroxide Magnetic Microparticles Capture Probe

Cobas Amplicor PCR Sensitivity 82 – 98%, specificity >99% Test up to 66 patient specimens in one day on one COBAS Analyzer Swabs in Amplicor specimen transport medium store at RT for up to 10 days. Urine – store up to 24h at RT and up to 7 days at o C Internal amplification control Multiplex available to detect CT and GC

Cobas Amplicor Tecan MiniprepCobas

RNA transcription amplification system using two enzymes, reverse transcriptase and T7 RNA polymerase Isothermal, amplification of rRNA target Produces over ten billion-fold amplification Single-tube format Gen-Probe Aptima Combo 2 assay Transcription-Mediated Amplification (TMA)

Gen-Probe APTIMA Combo 2 Test Detects Chlamydia and Neisseria gonorrhoea Uses three technologies: Target capture specimen processing Transcription-Mediated Amplification Dual Kinetic Assay (DKA) detection

Target Capture Technology Designed to: Reduce false negatives by removing inhibitors Simplify sample processing Different specimens Allows testing of urine and swabs in same run

Transcription-Mediated Amplification

Detection by Dual Kinetic Assay (DKA) Technology Modification of Hybridization Protection Assay (HPA) Technology Two different acridinium ester labels on different DNA probes Allows simultaneous detection of two different nucleic acid targets

Hybridization Protection Assay Hybrids Hybridization rRNA DNA Probe AE 60°C 1 hour Hybridize +

Selection/Detection Hybridization Protection Assay luminometer

Gen-Probe Aptima-Combo 2 Assay Sensitivity 94 – 100%, specificity 98 – 100% One sample, One test = Two results CT/GC targets co-amplified and individually detected in a single tube Suitable for large workloads Transport medium allows storage up to 30  C: 30 days for urine; 60 days for swabs

Automation with the DTS 1600 Instrument

Tigris In development Full automation: sample processing, amplification, detection Continuous specimen loading capability Simultaneous analysis of up to four different assays

ProbeTec Strand Displacement Amplification (SDA) Target gene – chlamydia plasmid 1.Target generation phase dsDNA heat denatured to give ssDNA 2.Amplification phase SDA isothermal. ssDNA amplified using DNA polymerase, restriction enzyme, primers with restriction enzyme recognition sites, bumpers 3. Detection phase – ET fluorescence energy transfer

ProbeTec SDA ssDNA with restriction siteAmplification primer binds DNA polymerase extends primerdsDNA with restriction sites

ProbeTec SDA Restriction enzyme binds Nicks one strand dsDNA DNA polymerase binds, extends displacing previously made strand Restriction site repeatedly nicked and new strands made and displaced Each displaced strand enters cycleDNA strands bind to probe and detected by Energy Transfer Hairpin secondary structure Fluorescent dye Quenching dye

ProbeTec ET Assay

Sensitivity 80.5 – 95.7%, Lower sensitivity for female urines Specificity 93.8 – 99.8% Internal amplification control Rapid turnaround Suitable for large workloads Semi-automated Viper

Near Patient Tests EIA based available Result in 30 minutes Less sensitive and specific Higher cost NAAT based tests in development More sensitive and specific but high costs

Chlamydia trachomatis Which Test for Screening?

Ideal Screening Test High sensitivity and specificity Suitable for non invasive samples – urine Fast turnaround time Low cost Not affected by inhibitory substances Transport and storage of samples not critical

Ideal Screening Test Does not require expensive equipment, expertise or separate work areas Suitable for large workloads Able to automate Easy to perform – “black box” Point of care testing – increase compliance

We believe it is scandalous that a sub-optimal test, with an accuracy rate markedly below the best tests, is still widely in use in England for the detection of chlamydia. Indeed, we believe that health providers would be highly vulnerable to damages claims made by patients who had received a false negative diagnosis and had thus not had treatment for chlamydia infection. House of Commons Select Committee on Health - Third Report 22 May 2003 Which Test?

Cell culture, DIF, EIA, hybridisation assays not sensitive enough. NAATs higher sensitivity (urine?) Which NAAT? – depend on local needs but how do they compare in performance? DoH funded study in collaboration with MHRA ( Medicines and Healthcare products Regulatory Agency) and MiDAS ( Microbiological Diagnostics Assessment Service) to evaluate three NAATs

Evaluation of NAATs Aim to provide information on: Comparative performance of three commercial NAATs using urine samples Which ones best meet the needs of Trusts and the national screening programme Results from repeat testing – testing algorithm Development of reference testing algorithm and role of Lightcycler assays Future evaluation design and logistics

NAAT Evaluation Three evaluation sites: TMA – Liverpool - Aptima Combo/DTS 1600 SDA – Portsmouth - Probetec ET with Viper? PCR - UCL/Liverpool - Cobas Amplicor/Miniprep Urine samples submitted for routine testing Sample anonymised and divided in 4 aliquots Samples sent overnight to other labs for testing Each site neg and 170 pos (M/F) Total 1530 neg and 510 pos tested by 3 methods Aim to detect difference in sensitivity of 5%

Portsmouth Routine samples SDA 170 positive/510 negative Four aliquots TMA Liverpool PCR UCLH SDA Portsmouth PCR Liverpool/UCLH 170 positive 510 negative TMA Liverpool 170 positive 510 negative STRBL repeat next day

Developments Full automation Combined chlamydia/gonorrhoea test New molecular tests e.g Abbott and molecular beacons to replace LCx Combined cervical cytology and chlamydia sample Point of care NAAT – portable “black box” Screening of men?

Acknowledgements Roche Diagnostics Gen-Probe Becton Dickinson