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Chlamydia NAATs: update in the clinical and laboratory setting

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Chlamydia NAATs: update in the clinical and laboratory setting Gill Underhill St Mary s Hospital Portsmouth Chlamydia trachomatis Chlamydia trachomatis Commonest ... – PowerPoint PPT presentation

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Title: Chlamydia NAATs: update in the clinical and laboratory setting


1
Chlamydia NAATs update in the clinical and
laboratory setting
  • Gill Underhill
  • St Marys Hospital
  • Portsmouth

2
Chlamydia trachomatis
Obligate intracellular gram negative organism
 
Two forms Infectious EB elementary body
spore like Replicating RB reticulate body
metabolically active
  •  
  •  

From www.chlamydiae.com
3
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

4
Chlamydia Infection in Men
  • Asymptomatic infection 50
  • Non specific urethritis
  • Strong associations with
  • Acute epididymitis
  • Prostatitis
  • Male infertility

5
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 lt 20 have infertility
  • Fitz-Hugh-Curtis syndrome peri-hepatitis
  • Peri-splenitis, peri-nephritis, peri-appendicitis

6
Other Diseases
  • Proctitis especially homosexual men
  • Reactive arthritis acute onset. Involves mostly
    knees, ankles, toes
  • Reiters syndrome mainly men - iritis, uveitis,
    conjunctivitis, and urethritis
  • Neonates infected during birth
  • 20 conjunctivitis and/or pneumonia

7
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

8
Diagnostic Options for Chlamydial Infections
  • Cell Culture
  • Direct Fluorescence Assay
  • Enzyme Immunoassay
  • Hybridisation Assay
  • Nucleic Acid Amplification Tests

9
Cell Culture
  • 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

10
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

11
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

12
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

13
Nucleic Acid Amplification Tests
  • Technically demanding
  • Special areas required
  • Contamination problems
  • Specimen transport and storage critical
  • Expensive
  • No confirmatory test
  • PCR, SDA, TMA, (LCR)

14
Polymerase Chain Reaction
Roche Amplicor first commercially available PCR.
Target is chlamydia plasmid. Primers are biotin
labelled to produce biotin labelled amplicons
15
Cobas Amplicor Denaturation Hybridization
16
Cobas Amplicor Conjugate Substrate Addition
17
Cobas Amplicor PCR
  • Sensitivity 82 98, specificity gt99
  • 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

18
Cobas Amplicor
  • Tecan Miniprep Cobas

19
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

20
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

21
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

22
Transcription-Mediated Amplification
23
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

24
Hybridization Protection Assay
Hybridization
rRNA
AE
60C

1 hour Hybridize
AE
AE
DNA Probe
AE
AE
Hybrids
25
Hybridization Protection Assay
Selection/Detection
luminometer
26
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

27

Automation with the DTS 1600 Instrument
28
Tigris
  • In development
  • Full automation sample processing,
    amplification, detection
  • Continuous specimen loading capability
  • Simultaneous analysis of up to four different
    assays

29
ProbeTec Strand Displacement Amplification (SDA)
  • Target gene chlamydia plasmid
  • 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

30
ProbeTec SDA
ssDNA with restriction site
Amplification primer binds
DNA polymerase extends primer
dsDNA with restriction sites
31
ProbeTec SDA
DNA polymerase binds, extends displacing
previously made strand
Restriction enzyme binds
Nicks one strand dsDNA
Hairpin secondary structure
Quenching dye
Fluorescent dye
Restriction site repeatedly nicked and new
strands made and displaced
Each displaced strand enters cycle
DNA strands bind to probe and detected by Energy
Transfer
32
ProbeTec ET Assay
33
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

34
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

35
Chlamydia trachomatis
  • Which Test for Screening?

36
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

37
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

38
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.

39
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

40
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

41
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

42
Portsmouth
Routine samples SDA 170 positive/510 negative
repeat
Four aliquots
SDA Portsmouth
STRBL
nextday
TMA Liverpool
PCR UCLH
PCR Liverpool/UCLH 170 positive 510 negative
TMA Liverpool 170 positive 510 negative
43
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?

44
Acknowledgements
  • Roche Diagnostics
  • Gen-Probe
  • Becton Dickinson
  • www.chlamydiae.com
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