CDC National Infertility Prevention Project
Laboratory Update
Region I
Wells Beach, Maine
June 1-3, 2009
Richard Steece, Ph.D., D(ABMM)
DrRSteece@aol.com
Chlamydia and Gonorrhea “102”
•Tests - Old and New
•Test Performance Issues
•Sensitivity/Specificity
•Positive Predictive Value (PPV)
•Negative Predictive Value (NPV)
•Frequently Asked Questions (FAQ)
Chlamydia
Laboratory Methods
• Culture (Cell Culture)
Advantages/Disadvantages
Cell Culture
ADVANTAGES
DISADVANTAGES
• Used for many types of
specimens, e.g.
endocervical, urethral,
rectal, ocular, etc.
• Meets medico-legal
standards (specificity)
• Used for strain studies
(DNA fingerprinting)
• Susceptibility testing
possible
• Comparatively expensive
• Many variables involved,
e.g. cell culture, medium,
etc.
• Technically more difficult
than many non-culture
tests
• Delayed turn around time
• Lack of sensitivity
(compared to amplified
tests)
Chlamydia Laboratory Methods
Non-culture
ANTIGEN DETECTION
Direct Fluorescent Antibody (DFA)
Enzyme Immunoassay (EIA)
NUCLEIC ACID DETECTION METHODS
Nucleic Acid Probe (GenProbe)
Hybrid Capture (Digene)
Advantages/Disadvantages
Non-culture
ADVANTAGES
DISADVANTAGES
• Used for many types of
specimens, e.g.
endocervical, urethral,
rectal, ocular, etc. (DFA)
• Effective for large scale
screening (EIA/NAP)
• Viable organism not
required
• Evaluate quality of
specimens (DFA)
• Inexpensive
• Rapid turn around time
• Not suitable for large
volume testing (DFA)
• Lack of sensitivity
(compared to amplified
tests)
• Not FDA cleared for
alternate specimens, e.g.
urine, etc.
Chlamydia Laboratory Methods
Non-culture
• Nucleic Acid Amplification Tests (NAATs)
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•
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Roche AMPLICOR® CT/NG Test (PCR)
Roche COBAS AMPLICOR™ CT/NG Test (PCR)
GenProbe APTIMA COMBO 2® (TMA)
GenProbe APTIMA CT® (TMA)
BD ProbeTec™ ET (SDA)
BD ProbeTec™ Chlamydia trachomatis (CT) Qx
Amplified DNA Assay (SDA)
• Abbott RealTime CT/NG (PCR)
Advantages/Disadvantages
NAATs
ADVANTAGES
DISADVANTAGES
• Most sensitive and specific tests
• Effective for large scale
screening
• Rapid turn around time
• NAATs are FDA cleared for urine
specimens
• May be used with some
alternate specimens
• Some versions not suitable
for large volume screening
• High technical skill
required
• Special facilities or clean
area required
• Expensive
Chlamydia Rapid Tests
Point of Care Tests (POCTs)
• Wampole Clearview
• Quidel Quickview - Inverness
Advantages/Disadvantages
POCTs
ADVANTAGES
DISADVANTAGES
• Rapid turn around
time
• Allows treatment of
patient while in clinic
• Expensive
• Not suitable for large volume
screening
• Poor sensitivity with some
POCTs
• Complexity non-waived
Gonorrhea
Laboratory Methods
Culture
• Thayer Martin, etc.
• Genetic transformation (Gonostat)
Direct Microscopic Exam
• Gram Stain
Advantages/Disadvantages
Bacterial Culture
ADVANTAGES
• All types of specimens,
e.g. endocervical,
urethral, rectal,
pharyngeal, ocular, etc.
• Meets medico-legal
standards (specificity)
• Used for strain studies
fingerprinting
• Susceptibility testing
possible
DISADVANTAGES
• Some variables, e.g. various
culture, media, etc.
• Delayed turn around time
• Lack of sensitivity
(compared to amplified
tests)
Gonorrhea
Laboratory Methods
Non-Culture
• Nucleic acid detection method
• Nucleic Acid Probe (GenProbe)
• Hybrid Capture (Digene)
Advantages/Disadvantages
NAP and NAPSA
ADVANTAGES
DISADVANTAGES
• Effective for large scale
screening
• Viable organism not
required
• Rapid turn around time
• Inexpensive
• Moderate technical skill
required
• Lack of sensitivity
(compared to amplified
tests)
• Not FDA cleared for
alternate specimens, e.g.
urine, etc.
Gonorrhea
Laboratory Methods
• Nucleic Acid Amplification Tests (NAATs)
•
•
•
•
•
•
Roche AMPLICOR® CT/NG Test (PCR)
Roche COBAS AMPLICOR™ CT/NG Test (PCR)
GenProbe APTIMA COMBO 2® (TMA)
GenProbe APTIMA GC® (TMA)
BD ProbeTec™ ET (SDA)
BD ProbeTec™ Neisseria gonorrhoeae (GC) Qx
Amplified DNA Assay (SDA)
• Abbott RealTime CT/NG (PCR)
Advantages/Disadvantages
Nucleic Acid Amplified Tests (NAATs)
ADVANTAGES
DISADVANTAGES
• Most sensitive and specific tests
• Effective for large scale
screening
• Rapid turn around time
• NAATs are FDA cleared for urine
specimens
• May be used with some
alternate specimens
• Some versions not
suitable for large volume
screening
• High technical skill
required
• Special facilities or clean
area required
• Expensive
Test Performance Issues
Sensitivity – The ability of a test to detect
patients who have the disease or
condition for which they are being tested
OR refers to the proportion of people with
disease who have a positive test.
Specificity – The ability of a test to
identify patients who do not have the
disease or condition for which they are
being tested OR refers to the proportion
of people without disease who have
negative test result.
• Positive Predictive Value (PPV)
– The likelihood that an individual
with a positive test has the disease.
• Negative Predictive Value (NPV)
– The likelihood that a person with a
negative test does not have the
disease.
Performance estimates1 of different tests to
detect chlamydia and gonorrhea
CT Culture
Sensitivity, %
40-70
Specificity,%
>99
GC Culture
45-65
>99
DFA
50-70
95-99
EIA
60-70
95-99
NAP/NAPSA
60-75
97-99
NAATs
95-98
>99
1Performance
estimates vary widely due to differences in statistical analysis
Example of NPV using Prevalence
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•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 20.0%
NPV=(1-.20)(.99)/(1-.20)(.99)+(.20)(1-.95)x100
NPV=.792/.792(.01)x100
NPV=.9875 x 100 = NPV of 99%
Example of NPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 20.0%
NPV=(1-.20)(.99)/(1-.20)(.99)+(.20)(1-.95)x100
NPV=.792/.792+(.01)x100
NPV=.9875 x 100 = NPV of 99%
• Sensitivity is 85%
• NPV=.9635 x 100 = NPV of 96%
Graph Courtesy of Abbot Laboratories
Example of PPV using Prevalence
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•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 20.0%
PPV=(.20)(.95)/(.20)(.95)+(1-.20)(1-.99)x100
PPV=.19/.19+(.008)x100
PPV=.9595 x 100 = PPV of 96%
Example of PPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 15.0%
PPV=(.15)(.95)/(.15)(.95)+(1-.15)(1-.99)x100
PPV=.1425/.1425+(.0085)x100
PPV=.9437 x 100 = PPV of 94%
Example of PPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 10.0%
PPV=(.10)(.95)/(.10)(.95)+(1-.10)(1-.99)x100
PPV=.095/.095+(.009)x100
PPV=.9134 x 100 = PPV of 91%
Example of PPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 5.0%
PPV=(.05)(.95)/(.05)(.95)+(1-.05)(1-.99)x100
PPV=.0475/.0475+(.0095)x100
PPV=.8333 x 100 = PPV of 83%
Example of PPV using Prevalence
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•
•
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Sensitivity is 95%
Specificity is 99%
Prevalence is 2.0%
PPV=(.02)(.95)/(.02)(.95)+(1-.02)(1-.99)x100
PPV=.019/.019+(.0098)x100
PPV=.6597 x 100 = PPV of 66%
Example of PPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99%
Prevalence is 1.0%
PPV=(.01)(.95)/(.01)(.95)+(1-.01)(1-.99)x100
PPV=.0095/.0095+(.0099)x100
PPV=.4896 x 100 = PPV of 49%
Example of PPV using Prevalence
(Effect of repeating positive specimens)
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1-(1-Spec.)2 = 1-(1-.99)2 = 1-(.0001) = 99.99%
Sensitivity remains 95%
Revised Specificity is 99.99%
Prevalence is 1.0%
• PPV=(.01)(.95)/(.01)(.95)+(1-.01)(1-.9999) x 100
• PPV=.0095/.0095+(.000099) x 100
• PPV=.9896 x 100 = PPV of 99% (49%)
Graph Courtesy of Abbot Laboratories
Example of PPV using Prevalence
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•
Sensitivity is 95%
Specificity is 99.9%
Prevalence is 1.0%
PPV=(.01)(.95)/(.01)(.95)+(1-.01)(1-.999)x100
PPV=.0095/.0095+(.00099)x100
PPV=.9056 x 100 = PPV of 91%
Example of PPV using Prevalence
•
•
•
•
•
•
Sensitivity is 95%
Specificity is 99.9%
Prevalence is 1.0%
PPV=(.01)(.95)/(.01)(.95)+(1-.01)(1-.999)x100
PPV=.0095/.0095+(.00099)x100
PPV=.9056 x 100 = PPV of 91%
• Specificity is 99.99%, PPV of 99%
Frequently Asked Questions (FAQ)
Question: How long after a patient has successfully completed
appropriate antimicrobial therapy would you be able to detect antigen
in their specimen (e.g. false positive) due to residual CT or GC
DNA/RNA?
Answer: The current CDC Treatment (and Laboratory) Guidelines state
that DNA or RNA may persist for up to 3 weeks after the successful
completion of appropriate antimicrobial therapy. At the recent
laboratory guidelines meeting, the expert panel recommended that this
statement remain the same at this time. However, studies are being
conducted to examine this with several "newer" NAATs and it is
possible this recommendation may change in the future based on new
data.
Frequently Asked Questions (FAQ)
Question: How soon after a patient has unprotected sex would you
be able to detect CT and/or GC with a nucleic acid amplification test
(NAAT)?
Answer: Depending on the antigen load (i.e. amount of CT or GC in
the ejaculate), a patient’s specimen could be positive very shortly
after exposure (sex/rape/abuse), before actual infection, from
minutes to hours after sex to upwards of several days. If infection
occurs there should be a latent or silent (undetectable) period for a
short time. Once again depending on antigen load, a positive (if the
individual becomes infected) could be detected in some cases in as
little as a week, looking at the life cycle of the organism. However,
the average time from actual infection to detectable shedding is more
likely 2-4 weeks for CT, most likely a week sooner for GC.
Frequently Asked Questions (FAQ)
Question: Is test-of-cure recommended as a routine procedure after
therapy for CT or GC infection with first-line CDC-recommended
treatment regimens?
Answer: The guidelines do not recommend a test of cure for CT or
GC, with a few exceptions
The End - Questions
Lots of tears
Frequently Asked Questions (FAQ)
Question: In the past our laboratory has given us the option of
submitting urine or endocervical swabs on our patient/clients.
Recently they have verified rectal swabs as an additional specimens.
Is it possible to just send in rectal swabs instead of urine or
endocervical?
Answer: Rectal swabs are not recommended for patients unless they
have participated in anal sex.