Welcome to the AACC Expert Access Live Online program.

Our topic this month is is Rapid Testing for Streptococcal Infection: How Effective Is It? . The preceding 25 years have witnessed tremendous changes in the laboratory diagnosis of group A streptococcal infection. Whereas culture was once the mainstay for diagnosis, rapid antigen testing is now widely used in clinical laboratories. However, the ease of antigen detection testing and rapid results belie a critical shortcoming of these assays. Their lack of sensitivity compared to culture prompts most authorities, including the American Academy of Pediatrics and the Infectious Disease Society of America, to recommend culture confirmation of negative test results from antigen-based testing.

DNA-based detection of group A strep has been proposed as a way to overcome the lack of sensitivity exhibited by antigen testing, but the high initial cost of molecular assays have discouraged labs from considering them as an option for routine strep testing. Today's expert is Franklin Cockerill, III, MD, Chair of the Division of Clinical Microbiology; Director, Bacteriology Laboratory; and Director, Syphilis and Helicobacter Serology Laboratory at the Mayo Clinic in Rochester, Minn. Dr. Cockerill's presentation outlines the pathophysiology of strep A infection and describes Mayo's experience in developing and implementing a DNA-based assay to replace the antigen/culture strep testing algorithm that was used for years. Is molecular strep testing an option for your lab? View the presentation and direct your questions to this month’s online expert, Dr. Franklin Cockerill, III.

AACC would like to extend its most sincere thanks to the Bayer Corporation for making this program possible.

I certainly agree with your rationale for dx of Strep A infection in primary care (treatment prevents sequelae). When you began looking to replace your antigen-based Strep testing program, were there any considerations other than improving the program's performance and detection rate? Did you look at overall cost, cost per test, cost per dx, or any other economic indices? Did your group make any estimates concerning $ saved by preventing sequelae?
Phoenix, AZ

Franklin R. Cockerill, III, MD: We are currently planning a well-designed clinical trial to assess the “bedside” economic factors associated with this rapid DNA-based testing method. We presume that a more rapid turn-around time for results should lessen the inappropriate use of antibiotics. A more rapid result should result in an improvement in symptoms more quickly and decrease the frequency of serious sequelae so that the patient returns to preschool, school or work sooner. A more rapid result should lesson the spread of streptococci to others. Our economic team working on the design of this study hope to be able to evaluate these outcomes which should be not only be of personal medical benefit to the patient but also should be cost saving. We also encourage other investigators to conduct similar cost-effectiveness trials. As shown in the PowerPoint presentation, slide 60, entitled, “Group A Streptococcus LightCycler Assay vs. Antigen/Culture,” the personnel time for the LightCycler method was less than half the time required for the Antigen/Culture (three minutes vs. seven minutes). We conduct approximately 30,000 tests per year at our institution. Combining the cost of reagents and the cost of the LightCycler instrument (about $50K depreciated over three years), and the savings in personnel costs, it is cheaper for us to perform the LightCycler assay versus the Antigen/Culture assays. For smaller operations, fewer savings may be realized, but the LightCycler instrument can be used for a variety of other assays, which should contribute to cost-effectiveness. We currently are running 12 LightCycler assays for a number of different pathogens on 10 LightCyclers.

In regard to the declining sensitivity of the rapid antigen assays, do you think this is a function of declining manufacturing standards, the different mix of strep variants among different hospitals, or the result of some other factor?
Boston, Mass.

Franklin R. Cockerill, III, MD: We share your assumptions about the declining sensitivity of rapid antigen assays for detecting Group streptococci. We wonder whether the antigen measured by these may be expressed in different amounts in different streptococcal strains. Accessibility of antibodies in immunoassays to these cell-wall antigens (we presume assays are measuring Lancefield Group A carbohydrate antigen) may also be compromised due to varying thickness of the hyaluronic acid capsule that surrounds the Group A streptococcus. Finally, we wonder whether various companies that produce these immunoassays use a common source of antibody (e.g., the same rabbit colony). We have advised several companies that produce these immunoassays and the FDA about our findings of decreased sensitivity of these assays.

What is the cost of DNA based Group A Stretococcus testing compared to antigen testing, and culture?
Philadelphia,PA

Franklin R. Cockerill, III, MD: As shown in the PowerPoint presentation, slide 60, entitled, “Group A Streptococcus LightCycler Assay vs. Antigen/Culture,” the personnel time for the LightCycler method was less than half the time required for the Antigen/Culture (three minutes vs. seven minutes). We conduct approximately 30,000 tests per year at our institution. Combining the cost of reagents and the cost of the LightCycler instrument (about $50K depreciated over three years), and the savings in personnel costs, it is cheaper for us to perform the LightCycler assay versus the Antigen/Culture assays. Reagents for the Strep A Assay are available from Roche. For smaller operations, fewer savings may be realized, but the LightCycler instrument can be used for a variety of other assays, which should contribute to cost-effectiveness. We currently are running 12 LightCycler assays for a number of different pathogens on 10 LightCyclers.

Just a comment that it's taking forever for the photos on the slides to come through -- and I've got a mega internet connection.
Spokane, WA

Franklin R. Cockerill, III, MD: Sorry, I included a fair number of images and therefore probably the reason for the delay.

What is known about the decline in the sensitivity of rapid antigen tests? The online presentation does not indicate whether or not the reason is thought to be one of personnel competence or one of gentic or antigenic drift over time, or some other factor. I'd appreciate your thoughts on this.
Spokane, WA

Franklin R. Cockerill, III, MD: We can only speculate about the declining sensitivity of rapid antigen assays for detecting Group streptococci at our institution. We wonder whether the antigen measured by these may be expressed in different amounts in different (newly emerging) streptococcal strains. Accessibility of antibodies in immunoassays to these cell-wall antigens (we presume assays are measuring Lancefield Group A carbohydrate antigen) may also be compromised due to varying thickness of the hyaluronic acid capsule that surrounds the Group A streptococcus. Finally, we wonder whether various companies that produce these immunoassays use a common source of antibody (e.g., the same rabbit colony). We have advised several companies that produce these immunoassays and the FDA about our findings of decreased sensitivity of these assays.

The rapid antigen test is a realy rapid test. Test result can be obtained with 10 minutes. Further more, it is a cost-effective test. This test is particular suitable for Point-of Care. How about a combination of rapid antigen test and your rapid-PCR test?
San Diego

Franklin R. Cockerill, III, MD: Combining the rapid antigen test with the rapid PCR test in the same way as the conventional approach for detection of Group A streptococci from throat swabs (i.e., rapid antigen and if negative followed by culture) could be done. One has to remember that the advantage of doing the PCR test as a stand-alone test is that it saves time for the patient in the long run. As an example, in February-March, we have a frequency of 25% true-positives. If the antigen assay is 70% sensitive, 13 of 25 patients out of 100 patients will have a positive result by the antigen assay. The remaining 87 patients have to wait up to 48hr for a confirmatory result by culture. Another point, the combination of rapid antigen testing and PCR testing would add to the cost. We batch our PCR tests in 4-6hr runs, which assures that the latest a test result is available to the patient is 8 hours. Our patients have accepted this with great enthusiasm. The turn-around time is much better than the traditional approach. Our patients leave the clinic and call an 800 number to get the result. An automated message informs them the result and if positive advises where the patient can pick up a prescription. Please note that I should have used the figure 50% instead of 70% above.

What were your primer pairs used for the realtime PCR?
New Orleans

Franklin R. Cockerill, III, MD: Mayo has licensed the primer and probe pairs for this test to Roche which has introduced them as ASRs. If anyone wants primer and probe sequences for research purposes Mayo will provide these with a MTA (Materials Transfer Agreement). We welcome additional clinical studies with this methodology.

Does Strep A exist in saliva and if so what are the concentrations in saliva?
Pleasanton, CA

Franklin R. Cockerill, III, MD: Interesting question. There is some old literature (I believe one of the authors is Edward Kaplan) that evaluated the recovery by culture of Group A streptococcus from saliva versus the conventional specimen, swabbed pharynx. As I recall, the best method for recovery was the swab specimen, but saliva does contain the organism. I do not know if concentration of the organism in saliva was assessed, but this would be a difficult task.

How does the PCR test compare in sensitivity to the 24 hour Lim broth biologically amplified then Gen Probe DNA probe confirmed test system for Group A strep? What are the comparative retail list prices for the PCR test versus Antigen with reflex to culture?
Baltimore, MD

Franklin R. Cockerill, III, MD: We have not performed a study comparing the GenProbe assay to this PCR assay but encourage that it be done. As part of the study one should also compare the personnel time involved as well as the cost of reagents and instrumentation. Mayo has licensed this Strep PCR assay to Roche and my understanding is that it is now available as an ASR in the United States. Therefore, anyone should be able to do the comparison study. Considering that the PCR test requires less than half the personnel time for antigen/culture method in our laboratory and the cost for the ASR and instrument, the total cost for performing the PCR test is less than the cost for performing the antigen/culture method.

Is this procedure one that is batched or are tests performed one at a time? If they are performed one at a time, can other tests be started before one is finished? Also, how does the cost of performing this test compare to antigen/culture and to reimbursement?
Columbus, Ohio

Franklin R. Cockerill, III, MD: We batch approximately every 4-6hr during the morning and evening shifts. The last batch is at 11PM with the first 7AM. This works best for our ER and outpatient clinics. This assures that all patients have a result at least by 8hr. One LightCycler run will accommodate 32 samples (30 specimens and 2 controls). We use the same settings (parameters) for all LightCycler assays so e.g. a Bordetella, Legionella, VRE, MRSA, Group A and Group B can be performed simultaneously. The instrument is not a continuous flow but because instrument time is short, this does not seem to be a factor. Please refer to slide 60 which shows that one half the personnel time is required for the PCR method. Considering our volume of 30,000 tests per year, personnel costs and reagent/instrument costs, the cost of performing the PCR test is less than the cost for performing the antigen/culture test at our institution.

Is this a "home brew" test? Are ASR being used?
Salem, Oregon

Franklin R. Cockerill, III, MD: Mayo has licensed our "homebrew" reagents to Roche. ASRs should be available from Roche.

I wanted to rephrase an earlier question submitted this morning. I had an error in one part of the question. The replacement question is... What is the comparative sensitivity of the new PCR Strep A test against the DNA Probe method that is marketed by Gen Probe? Thanks and sorry for the confusion with the first question submitted earlier today.
Baltimore, MD
Franklin R. Cockerill, III, MD
Please see my earlier response to your same question. A comparative study is required and should be doable since reagents for both methods should be available from GenProbe and Roche. In our experience, the GenProbe method was less sensitive than our standard culture method (Pokorski, Journal Clin Microbiol, vol 32, 1440-1443). The PCR test is as sensitive as our standard culture method. The GenProbe, in our experience, is also more hands-on.

Since beta strep Group C is also a major cause of pharyngitis, wouldn't it be better to develop a kit that checked for both Group A and Group C? If it only checks for Group A, we would still need to backup with a culture. Since this DNA test would be more costly than the current rapid tests, it would be nice to really eliminate some of the backup culture work that would need to be done to check for all pertinent groups.
Fresno, CA.

Franklin R. Cockerill, III, MD: Interestingly, by melting curve analysis, this PCR test will also pick up Group C and Group G Strep. So those who want to look for these strep can do so with the assay. Please see slide no. 56. The first curve is a broken line that one sees with both Group C and Group G Strep. This infomation will be detailed in a paper accepted for publication in the Journal of Clinical Microbiology.

Dear sir. I really fascinated by this test, and I would like to apply this test in my laboratory, which it is equiped with PCR equipments. From which supplier could I obtain the material and the method of the test.?? Thank you and best regards.
Aleppo, Syria

Franklin R. Cockerill, III, MD: Roche. Mayo has licensed the test to Roche, which also is the company that sells the LightCycler.

What swab system do you use to collect and transport the throat samples to the lab? How do you process the swab sample to extract the DNA once the swab is received in the lab?
Columbus, Ohio

Franklin R. Cockerill, III, MD: Any of a variety of swabs can be used. The extraction is performed using a S.E.T.S. (Swab Extraction Tube System) tube. We developed this extraction tube in our laboratory and Mayo has licensed this to Roche. The extraction process takes only minutes.

What kind of sample processing needs to be performed prior to loading the samples into the Lightcyler? How long does this take?
San Diego, CA

Franklin R. Cockerill, III, MD: A SETS (Swab Extraction Tube System) was developed in our lab which permits DNA extraction from the swab in minutes. Mayo has licensed this to Roche which should be available along with the ASRs.

Has the study on the detection sensitivity of the 8 immunoassays been published? If so, please provide the reference. Thanks.
Lexington, Kentucky

Franklin R. Cockerill, III, MD: No, it has not.

Please discuss the rationale behind the recommendation by the IDSA for ignoring the 10% culture positive pediatric patients who are missed by rapid AG testing. Is there a decline in non-suppurative post-streptococcal complications so ignoring this group is OK?
Kansas City, Mo.

Franklin R. Cockerill, III, MD: My interpretation of the IDSA guidelines is that a culture back-up is recommended for negative antigen tests for chilren but not adults. This is because non-suppurative complications are less frequent in adults. I would argue that the same requirements should hold for adults as well as children. Inappropriate antibiotic use would be lessened and adults who might spread the strep to others would be appropriately treated. There are good studies that show that early treatment of Group A strep results in resolution of symptoms and pharyngeal carriage sooner.

Dr. Cockerill, Could you comment on the test cost vs. reimbursement for the GAS PCR assay. In addition, I understand that a GBS assay is imminent. Can you comment on that assay yet and what it's impact will be considering the new guidelines surrounding GBS? Thanks, Jim Ivey.
Flint, MI

Franklin R. Cockerill, III, MD: If one uses the Medicare reimbursement rate for molecular-genetic based CPT codes for Group A strep and considers the cost of reagents, instrumentation, in our practice,there still is a margin. We have set the charge for our test as very comparable to the conventional antigen/culture method. Yes, we have arrangements like the Group A strep test to license a Group B test to Roche and anticipate that should be available early next year. That should be a very exciting test as we should be able to decrease the turn-around time considerably. For that test we use a proprietary stool buffer that enhanses sensivity and can be used directly on the MagNA Pure. Also, the Group A test, by melting curve analysis, can detect Group C and Group G strep, which also may cause pharyngitis.

Are you aware of any studies comparing Gen-Probe's direct assay with the Light Cycler protocol?
San Diego, CA

Franklin R. Cockerill, III, MD: No, these needs to be done. A previous study by our group (JCM Vol 32, 1440-1443) suggested that the GenProbe was not as sensitive as our standard culture method (the LightCycler method is. I haven't worked with any updates of the GenProbe method, but the version we used several years ago was manual and labor intense.

Hello from Mr. Dana Moses in Molecular Pathology! 1) How do you sell this test to busy ER doctors, who just want an answer, when your molecular lab is only open Mon-Fri, 7am-330pm? 2) Is the test easy enough for off-shifts to perform? 3) How doe one get started in developing rapid strep method (besides getting a LightCycler)? Thanks for your time...Dana
Hinsdale, Illinois

Franklin R. Cockerill, III, MD: We do the test in the routine lab at 4-6hr shifts with last batch around 11PM and first 7AM. This has worked very well with our ER and outpatient MDs. Patients call an 800 number for the result and and electronic voice tells them where to pick up a prescrition. All in all this process is much faster than old antigen/culure method. The test is nearly completely automated and so it can be performed in the routine laboratory. Mayo has licensed the reagents for this test to Roche. Otherwise, anyone can develop their own homebrew method, but from personal experience this will take time and trouble-shooting. Any rapid real-time platform/instrument could be used, which I would definitely recommend over traditional PCR methods.

Can the effectiveness of rapid antigen assays vary with population? I work at a University Health Center - primarily 18 - 25 year olds and we have used BioStar MAX for abotu 6 years. We quit doing back up cultures after a couple of years because we were not finding cases of strep being missed on the rapid. There was only one case that I can remember and it only grew one colony on culture when the rapid was negative. The only time we are doing cultures now is by request or if another pathogen is suspected. We do about 20-30 tests/day, but cannot imagine that the PCR test would be cost effective for us. Comments?
Bloomington, IN

Franklin R. Cockerill, III, MD: We evaluated the BioStar method (JCM Vol 32, 2698-2701) and found it to be considerably less sensitive than culture. I think that although many places believe they do well at picking Group A strep from a blood plate, they may miss them. This aside, total test volume, and the age of the patient (adults have less nonsupurative sequelae than children) may make it very reasonable in your setting to use the OIA method. Adults may transmit strep to kids and so a sensitive method for detection in adults may as an important consideration as in kids. The LightCycler method can be used to test for a variety of other pathogens...so using this platform for a vairiety of tests may be cost-effective in centers with lower volumes. We currently have 10 LightCyclers on which 11 different tests are performed.

Once you extract the swab in the SETS tube, how do you extract and prep the nucleic acid from the organism?
Columbus, Ohio

Franklin R. Cockerill, III, MD: The bacteria that are extracted from the swab using the SETS tube system are suspended in sterile water and then boiled. 32 samples can be prepared in about 15-20 minutes and then place in the LighCyler.

What is the appx cost?
Atlantic city, NJ

Franklin R. Cockerill, III, MD: The cost has been previously discussed. Based on reagents, instrument and personnel costs, and considering our volumes, this method is cheaper than our traditional antigen-culure method.

What is the cost difference in the gold standard and the new technology. Please include the savings that would be attained with a more rapid diagnosis. Is this something a routine Microbiology department could be doing or does it take special training, etc.
Fresno, Ca

Franklin R. Cockerill, III, MD: I believe the laboratory costs have been previously discussed in regards to the traditional antigen/culture method. Bedside cost savings should also be realized due to the high accuracy of the test and the decreased turn-around time for results. A proper cost-effective study is encouraged. The technology is nearly completely automated. No specialized training is necessary. Microbiology techs do all of our Strep, Bordetella LighCycler testing. Soon they will also be doing VRE and MRSA, Group B Strep, Verotoxin gene, C. difficile LightCycler testing. Virology techs do herpes simplex, varicella zoster, EBV, enterovirus testing which is the same technique.