1. Richard Steece, Ph.D., D(ABMM) DrRSteece@aol.com
CDC National Infertility Prevention Project Laboratory Update Region I Wells Beach, Maine June 1-3, 2009
Richard Steece, Ph.D., D(ABMM)

2. 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)

3. Chlamydia Laboratory Methods
Culture (Cell Culture)

4. Advantages/Disadvantages Cell Culture
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
DISADVANTAGES
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)

5. 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)

6. Advantages/Disadvantages Non-culture
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
DISADVANTAGES
Not suitable for large volume testing (DFA)
Lack of sensitivity (compared to amplified tests)
Not FDA cleared for alternate specimens, e.g. urine, etc.

7. Chlamydia Laboratory Methods Non-culture
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 CT® (TMA)
BD ProbeTec™ ET (SDA)
BD ProbeTec™ Chlamydia trachomatis (CT) Qx Amplified DNA Assay (SDA)
Abbott RealTime CT/NG (PCR)

8. Advantages/Disadvantages NAATs
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
DISADVANTAGES
Some versions not suitable for large volume screening
High technical skill required
Special facilities or clean area required
Expensive

9. Chlamydia Rapid Tests Point of Care Tests (POCTs)
Wampole Clearview
Quidel Quickview - Inverness

10. Advantages/Disadvantages POCTs
Rapid turn around time
Allows treatment of patient while in clinic
DISADVANTAGES
Expensive
Not suitable for large volume screening
Poor sensitivity with some POCTs
Complexity non-waived

11. Gonorrhea Laboratory Methods
Culture
Thayer Martin, etc.
Genetic transformation (Gonostat)
Direct Microscopic Exam
Gram Stain

12. Advantages/Disadvantages Bacterial Culture
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)

13. Gonorrhea Laboratory Methods
Non-Culture
Nucleic acid detection method
Nucleic Acid Probe (GenProbe)
Hybrid Capture (Digene)

14. Advantages/Disadvantages NAP and NAPSA
Effective for large scale screening
Viable organism not required
Rapid turn around time
Inexpensive
Moderate technical skill required
DISADVANTAGES
Lack of sensitivity (compared to amplified tests)
Not FDA cleared for alternate specimens, e.g. urine, etc.

15. 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)

16. Advantages/Disadvantages Nucleic Acid Amplified Tests (NAATs)
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
DISADVANTAGES
Some versions not suitable for large volume screening
High technical skill required
Special facilities or clean area required
Expensive

17. 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.

18. 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.

19. Performance estimates1 of different tests to detect chlamydia and gonorrhea
Sensitivity, % Specificity,%
CT Culture >99
GC Culture >99
DFA
EIA
NAP/NAPSA
NAATs >99
DNA amplified testing is most sensitive, New gold standard, can be used to test urine. Particularly important for screening males.
1Performance estimates vary widely due to differences in statistical analysis

21. 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%

22. 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%

23. Graph Courtesy of Abbot Laboratories

24. Example of PPV using Prevalence
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%

25. 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%

26. 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%

27. 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%

28. Example of PPV using Prevalence
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%

29. 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%

30. Example of PPV using Prevalence (Effect of repeating positive specimens)
Sensitivity remains 95%
Revised Specificity is 99.99%
Prevalence is 1.0%
PPV=(.01)(.95)/(.01)(.95)+(1-.01)( ) x 100
PPV=.0095/.0095+( ) x 100
PPV=.9896 x 100 = PPV of 99% (49%)

31. Graph Courtesy of Abbot Laboratories

32. 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%

33. 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%

34. 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.

35. 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. 

36. 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

37. The End - Questions
Lots of tears

38. 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.