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) 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 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)

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 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.

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)

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

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

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

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

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)

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

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.

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) 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

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 Sensitivity, % Specificity,% CT Culture 40-70 >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 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

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%

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 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 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) 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 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.