Nucleic Acid Amplification Tests for Chlamydia

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Nucleic Acid Amplification Tests for Chlamydia
Gonorrhea Reaping the Benefits and Avoiding the
Pitfalls

• Bobbie McDonald
• Advanced Microbiologist
• WSLH STD Program Coordinator
• May 26, 2004
• Bobbie_at_mail.slh.wisc.edu

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Epidemiology of CT GC

• Chlamydia
• Most commonly reported notifiable disease in US
• Over 800,000 cases reported to CDC in 2003
• 3 times more prevalent in women than men
• Most prevalent in females age 15-19
• Commonly asymptomatic

• Gonorrhea
• Second most commonly reported notifiable disease
• Over 350,000 cases reported to CDC in 2003
• Rates among men and women similar since 1997
• Most prevalent in women age 15-19, men 20-24
• Can be asymptomatic

• Both can lead to PID, ectopic pregnancy,
  tubal infertility

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Rates by sex U.S., 19812002 and the Healthy
People 2010 objective
Gonorrhea
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Gonorrhea Control

• A national gonorrhea control program was
  implemented in the mid-1970s
• From 1975-1997, rates dropped 73.8
• Slight increases in 1998 1999 have been
  followed by decreases
• Changes in screening practices and methods as
  well as reporting may contribute
• Gonorrhea Isolates Surveillance Project (GISP)
  monitors drug resistance

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Rates by sex United States, 19842002
Chlamydia
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Chlamydia Control Programs

• IPP demonstration project in HHS Region X in
  1988 other regions added in 93, 95
• Effectiveness of large-scale screening programs
  in reducing Chlamydia prevalence in women has
  been shown in older regions.
• From 1988-2002, CT prevalence dropped 55.4, from
  13.0 to 5.8 in family planning clinics
• Increases seen in some regions probably due to
  continued expansion of screening
• Since 2000, CT reportable in all 50 states

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CT GC Testing in the U.S., 2002
CT and GC Tests
24 million tests each
Chlamydia Infections
Gonorrhea Infections
Reported 834,503 Estimate 3 million
Reported 351,836 Estimate 650,000
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Laboratory Methods for CT and GC
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Laboratory Methods Gonorrhea

• Culture
• Nucleic Acid Transformation
• Nucleic Acid Probe (NAP- Gen Probe)
• Hybrid Capture (Signal Amplification)
• NAAT
• PCR (Roche)
• LCR (Abbott)
• SDA (BD)
• TMA / Aptimia (Gen Probe)
• Others on the horizon

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Laboratory Methods Chlamydia

• Isolation in cell culture
• Antigen Detection
• DFA
• EIA
• Automated, single-tube, micro-well formats
• Rapid or Point of Care tests
• Nucleic Acid Probe (NAP- Gen Probe)
• Hybrid Capture (Signal Amplification)
• NAAT (same tests as for GC)

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Method Selection

• Obvious factor in many labs Performance
• Other important factors to consider
• Complexity/ practicality ease of use
• Throughput/ volume/ turnaround time
• Prevalence in target population (PPV etc.)
• Cost issues
• Looking at the big picture, the most sensitive
  test may not be the best choice in a particular
  setting

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Efficiency Models Test Selection

• Compare costs of testing and treatment algorithms
  in populations of varying prevalence to find
  optimal strategy
• Consider the strategys total cost, number and
  proportion of positives detected and treated,
  cost per positive
• Optimal method based on goal impact most
  infections within cost constraints

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Point of Care Testing

• Despite the relatively low sensitivity, use of
  rapid tests can actually result in more
  infections being treated
• Can be useful in settings where
• Loss of patients to follow-up is high
• Few patients are treated empirically
• Rapid tests should NOT be used if not done
  while the patient waits

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Selection of an NAAT for CT GC

• A variety of methods to choose from
• Many differences are primarily logistical
• Size of instrumentation, throughput, degree of
  separation required/number of areas, automation
  etc.
• Until recently, performance differences were
  generally considered to be minimal
• Second generation tests claim better
  sensitivity
• More published data will clarify this issue
  further

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Laboratory Implementation of NAAT for Chlamydia
and Gonorrhea
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Implementation of NAAT

• NAAT requires more attention to procedural detail
  and QC than non-NAAT methods
• High sensitivity, ease of contamination
• Susceptibility of reactions to inhibition
• Critical areas to address include
• Training (clinician and lab staff)
• Development of SOP and QA protocols
• CLIA issues

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Training Clinicians

• Should occur with any change in method, and
  address
• Indications, appropriate specimen types
• Instruction on collection of all specimens, with
  specifics on proper collection of endocervical
  swabs
• Storage and transport requirements
• Result Interpretation
• Should also include laboratory monitoring of
  collection and transport, and periodic retraining

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CLIA Requirements for NAAT

• Facility Administration (subpart J)
• Lab design separate areas for reagent and
  specimen preparation, amplification and detection
• Amplification procedures not in a closed system
  must maintain uni-directional workflow
• Quality Systems

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Implementation SOP Development

• Standard Operating Procedures are based on
  manufacturer product inserts and supplemental
  information
• NAAT SOPs should be sure to address
• Dedicated work areas and equipment, other
  procedures to minimize cross-contamination
• Inhibition rates and controls
• Appropriate QC procedures

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Quality Systems QA

• Quality Assurance is now Quality Assessment
• A mechanism to monitor and evaluate the overall
  quality of the testing process
• Goal is to ensure accurate, reliable results
• Involves all pre-analytic, analytic and
  post-analytic processes

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Quality Systems QC

• Quality Control positive and negative controls
  on each test run
• Additional positive control external to the kit
• Stock culture or known clinical specimen
• Extraction control necessary if method has an
  extraction step
• System to alert when positivity patterns or
  numbers are our of the norm

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Verification of Performance Specifications

• Ensure that the test performs in your laboratory
  as claimed by manufacturer
• Requires evaluation by testing of known positive
  and negative specimens
• Requirements are more extensive for non-FDA
  cleared methods
• See the CDC document Screening Tests To Detect
  Chlamydia trachomatis and Neisseria gonorrhoeae
  Infections 2002. MMWR 200251(No.
  RR-15)Appendix C

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Expected Impacts, Advantages and Potential
Pitfalls of NAAT
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Expected Impact of NAAT

• Increased sensitivity
• Magnitude of increase dependant on population,
  initial positivity rate
• Larger gains can be seen in lower prevalence
• More follow-up required
• Treatment / counseling
• Contact tracing, testing
• Increases in GC screening due to convenience
• May be a concern if prevalence is low

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Changes in CT Positivity, EIA to NAAT, Wisconsin
Family Planning
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Other Advantages of NAAT

• Less-invasive or alternative specimens
• Urine testing can broaden screening
• Female and male urine for both CT GC
• Female urine sensitivity may be slightly less
  than cervical swabs, but higher than other
  methods
• Self-collected vaginal swabs (Gen Probe) offer
  advantage of urine, easier handling
• Ability to test Thin Prep Pap specimen remnants
  for CT and GC (PCR)

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Potential Disadvantages/Pitfalls

• CONTAMINATION
• Because of the high sensitivity of NAAT,
  contamination of specimens, reagents or work
  surfaces with even small amounts of target or
  amplified material can cause false-positive
  results

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Contamination

• Not a question of IF but of WHEN
• Critical concern is how quickly a contamination
  event is recognized and what is done to resolve
  it
• QC monitors to detect potential problems
• Standard negative controls not enough
• Monthly environmental sampling not designed for
  quick response to events, but to detect ongoing
  procedural problems

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Detecting Contamination Events

• Potential monitors include
• Positivity overall and among subgroups
• Unusual or unlikely patterns of positivity
• Proportion of specimens in a gray zone or
  high-negatives and low-positives
• Reduced reproducibility of results
• Thresholds may differ between labs
• Exceeding threshold may only indicate that a
  closer look at all data is needed

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Identify Contamination Source

• Environmental sampling, pre- post- bleach
• Identify source target or product?
• Pinpoint beginning of event (time/location)
• Examine every step, even the unlikely
• Minute events that would be insignificant with
  conventional methods may have major impact
• Product contamination difficult to eradicate
• Air flow within lab space
• Tracking/ spreading on unlikely objects

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Contamination Aftermath

• The faster an event is detected, the faster the
  recovery
• When in doubt, DONT report it out
• Even if QC passed, if something does not look
  right during an event, dont report results
  (positives)
• Some specimens will need to be recollected.
• Replacing swabs with urine softens the impact
• Honesty about the situation builds trust
• Opportunity to educate/communicate encourage
  clinicians to discuss results they question

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Other Potential Pitfalls

• False-positives due to
• Low PPV (low prevalence, increased GC screening)
• Test of cure (too soon after treatment)
• GC cross-reactivity with other Neisseria species
• Overconfidence in results
• Loss of ability to monitor drug resistance
• Inability to test non-genital sites

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Other Potential Pitfalls

• False-positives (not contamination related)
• Care must be taken when interpreting any result
• Adding GC because its cheap or easy can lead to
  problems
• If TOC is done, wait at least 3 weeks
• Overconfidence in results because its DNA
• No test is 100 sensitive or specific (not even
  culture)
• Loss of ability to monitor drug resistance
• Significant with GC, less with CT (but still on
  the radar screen)
• Inability to test non-genital sites
• Need for oral, rectal screening tests not clear
• NAAT can be verified for these but can be
  difficult (low positivity)

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NAAT Issues To Resolve

• Confirmatory testing (low PPV)
• Antimicrobial resistance
• Amplification/ inhibition controls
• Forensic use (sexual abuse/assault)
• Specimen adequacy
• Alternate specimens

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Positive Predictive Value
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Confirmatory Testing for NAAT

• From CDCs 2002 Laboratory Guidelines
• All positive screening tests should be considered
  presumptive evidence of infection.
• An additional test should be considered after a
  positive screening test if a false-positive would
  result in substantial adverse medical, social, or
  psychological impact for a patient.
• Consideration should be given to routinely
  performing an additional test after a positive
  screening test if the PPV is low (e.g., lt90).
• Patients should be counseled regarding prompt
  treatment after a positive screening test because
  an additional test might be falsely negative.

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Confirmatory Options for NAAT

• Test a second specimen with a different test that
  uses a different target, antigen or phenotype,
  and a different format.
• Test the original specimen with a different test
  that uses a different target, antigen, or
  phenotype and a different format.
• Repeat the original test on the original specimen
  with a competitive probe.
• Repeat original test on the original specimen.
• Cannot use non-amplified tests to confirm NAAT

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Practical Confirmation of NAAT

• Cannot use non-amplified tests to confirm NAAT
  results
• Collection of a second specimen can be
  problematic, expensive
• Few labs have the resources to maintain a second
  NAAT platform for retesting
• Manufacturers are beginning to address need for a
  practical confirmation method that meets criteria
  Aptima ASR)
• Can increase PPV by selective screening

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Antimicrobial Resistance, GC

• 18.0 of isolates collected in 2002 by the
  Gonococcal Isolate Surveillance Project were
  resistant to penicillin, tetracycline, or both
• Resistance to Cipro was identified by GISP in
  1991 has been spreading eastward from HI and CA
  since 2001
• Cephalosporin resistance has not yet been
  identified in GISP
• Multi-drug resistant isolates and isolated with
  elevated MIC to azithromycin have been seen

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Amplification Control

• Tests each specimen for substances that inhibit
  amplification reactions
• If amplification is inhibited in the control, a
  negative result may be unreliable
• Use can reduce throughput, adversely impact
  clinics (most will be negative)
• Inhibition rates vary between labs and specimen
  types, as well as over time
• Use dictated by measured rates monitoring
  inhibition rates is recommended

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Sexual Assault and Abuse

• CT and GC testing serves 2 purposes
• Patient management requires high sensitivity
• Forensic evidence requires high specificity
• Culture is the recommended method for both CT and
  GC in abuse/assault cases
• Data and experience with NAAT is insufficient to
  assess applicability in such cases
• Some researchers suggest that NAAT for CT might
  be used if culture is unavailable, and if
  positive results are confirmed using 2nd NAAT
  targeting a different sequence

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Specimen Adequacy

• Despite the high sensitivity of NAAT, infections
  can be missed due to poor specimen collection
• Routine or periodic assessment of endocervical
  specimen quality is recommended for all types of
  CT tests (CDC)
• gt10 of specimens collected for C. trachomatis
  testing are probably unsatisfactory because they
  contain secretions or exudate, but lack
  endocervical cells
• specimens lacking these cells may yield reduced
  positivity

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Alternative Specimens

• Specimens such as self-collected vaginal swabs
  and residual liquid Pap specimens have been
  approved for use with some CT-GC NAATs (check
  with manufacturer)
• For rectal and pharyngeal specimens, culture for
  CT and GC is the only option
• Verification of NAAT for some alternative
  specimen types may be possible

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Summary Pros and Cons of NAAT

• NAATs are the most sensitive and specific tests
  for CT and GC
• Increased sensitivity can result in
• Broader availability of screening (urine etc)
• More infections detected, treated, prevented
• Tradeoffs include higher cost, increased
  complexity in the lab, loss of isolates and
  ability to test non-genital specimens

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NAAT Future Developments

• Though the technology is inherently expensive,
  costs can come down
• Competition (more tests to choose from)
• Automation, instrumentation to save labor
• Improved sensitivity, specificity, contamination
  control
• Built-in confirmatory assays (alternate target)
• Additional specimen types
• Improved specimen transport requirements

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References

• Centers for Disease Control and Prevention.
  Screening Tests To Detect Chlamydia trachomatis
  and Neisseria gonorrhoeae Infections 2002. MMWR
  200251(No. RR-15)
• Roberta A. McDonald and John Pfister. Effects of
  the Transition to Chlamydia Nucleic Acid
  Amplification Testing in a Public Health
  Screening Program. Oral Presentation at the
  National STD Prevention Conference, San Diego,
  CA, March 4-7, 2002.
• THOMAS L. GIFT, CATHLEEN WALSH, ANNE HADDIX, and
  KATHLEEN L. IRWIN, MD, MPH. A Cost-Effectiveness
  Evaluation of Testing and Treatment of Chlamydia
  trachomatis Infection Among Asymptomatic Women
  Infected With Neisseria gonorrhoeae. Sexually
  Transmitted Diseases ? September 2002
• GUOYU TAO, THOMAS L. GIFT, CATHLEEN M. WALSH,
  KATHLEEN L. IRWIN, and WILLIAM J. KASSLER.
  Optimal Resource Allocation for Curing Chlamydia
  trachomatis Infection Among Asymptomatic Women at
  Clinics Operating on a Fixed Budget. Sexually
  Transmitted Diseases ? November 2002