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
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Replicating
RB – reticulate body – metabolically active
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From www.chlamydiae.com
Chlamydia trachomatis
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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
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Asymptomatic infection ~ 50%
Non specific urethritis
Strong associations with:
Acute epididymitis
Prostatitis
Male infertility
Chlamydia Infection in Women
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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
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Cell Culture
Direct Fluorescence Assay
Enzyme Immunoassay
Hybridisation Assay
• Nucleic Acid Amplification Tests
Cell Culture
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Original gold standard
High specificity ~100%, sensitivity 65-80%.
Continuous cell lines - McCoy
Incubate 48 - 72 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
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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
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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
A-Tube
Biotin
Magnetic
Microparticle
D-cup
Capture Probe
Wash Wheel
Cobas Amplicor
Conjugate & Substrate Addition
Avidin-horseradish Peroxidase Conjugat
forming complex with Biotin
Tetramethylbenzidine
(TMB) Substrate
Hydrogen
Peroxide
Capture Probe
Magnetic Microparticles
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 2 - 8 oC
• Internal amplification control
• Multiplex available to detect CT and GC
Cobas Amplicor
Tecan Miniprep
Cobas
Transcription-Mediated
Amplification (TMA)
• 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
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
Hybridization
rRNA
AE
60°C
+
AE
DNA
Probe
AE
AE
1 hour
Hybridize
AE
Hybrids
Hybridization Protection Assay
Selection/Detection
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 site
DNA polymerase extends primer
Amplification primer binds
dsDNA with restriction sites
ProbeTec SDA
Restriction enzyme binds
Nicks one strand dsDNA
DNA polymerase binds, extends
displacing previously made strand
Hairpin
secondary
structure
Fluorescent
dye
Restriction site repeatedly nicked Each displaced strand enters cycle
and new strands made and
displaced
Quenching
dye
DNA strands bind to probe and
detected by Energy Transfer
ProbeTec ET Assay
ProbeTec ET Assay
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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
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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
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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
Which Test?
House of Commons Select Committee
on Health - Third Report 22 May 2003
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.
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 - 510 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
repeat
STRBL
Four aliquots
next
day
SDA
Portsmouth
TMA
Liverpool
PCR
UCLH
PCR
TMA
Liverpool/UCLH
170 positive 510
negative
Liverpool
170 positive
510 negative
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
www.chlamydiae.com