History of Laboratory Testing for Sexually Transmitted Bacteria: From Old School to Next Gen

Friday, December 23, 2016 Published in On-Demand Spring 2012 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Sexually transmitted infections (STIs) are an unfortunate fact of life. Although at the beginning of the AIDS epidemic in the U.S., at least, the numbers of Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) temporarily dipped due to increased use of condoms, they seem to be resurging. In northern Europe, CT is again prevalent.

Next-Generation Testing for CT/NG

Friday, December 23, 2016 Published in On-Demand Spring 2012 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Another major revolution in NG/CT testing is about to begin. The next generation nucleic acid amplification GeneXpert® CT/NG assay has been designed to be a more specific test for these sexually associated infectious disease agents than ever before. With its ease of use and ability to test one or as many samples as needed, this assay could expand the capability of NG and CT testing to virtually every laboratory, no matter how small or large and no matter what their demand. The age of send-out testing for these two STIs may soon be over.

From the Editor

Friday, December 23, 2016 Published in On-Demand Winter 2012 Written by David Persing M.D., Ph.D., Chief Medical and Technology Officer, Cepheid

During the production of this issue of On Demand, the CDC declared the official start to the flu season, the latest start in 25 years. Drs. Ellen Jo Baron and Fred Tenover have written an up-to-date review of molecular diagnostic methods for respiratory infections, including the Xpert Flu test which provides on-demand results for FluA/B and H1N1 novel 2009, all of which are circulating this flu season. As the science and practice of Precision Medicine evolves toward the ultimate goal — providing the right patients with the right doses of the right medication at the right time — the impact of molecular diagnostics has never been greater. In the article by Donna Wolk and Daniel Olsen, you will find an excellent overview of the Verification and Validation processes that underpin the basic credibility and reliability of the diagnostic results we furnish on a daily basis. We hope you will benefit from reading both articles in this issue.

Verification of Qualitative Real-time PCR Methods

Friday, December 23, 2016 Published in On-Demand Winter 2012 Written by Donna M. Wolk, MHA, Ph.D., D(ABMM) and Daniel Olson, MPH*

MEASURE TWICE, CUT ONCE ACCURACY.
Clinical laboratories rely on the FDA clearance/approval process to ensure that commercially available molecular tests have been evaluated and found to be accurate and medically useful. However, the FDA-approval process is only the first step towards ensuring diagnostic accuracy in your laboratory. The process of in-house verification is governed by the Clinical Laboratory Improvement Amendments (CLIA) regulations5, and begins with the laboratory undertaking studies to reproduce the manufacturer's claimed performance characteristics4,5,9. Verification of an FDA-cleared assay by a laboratory performing the test exactly as described in the product insert is less stringent than the validation process needed for a modified test. If a test is not performed according to the published manufacturer guidelines, for instance, if the sample transport matrix is different from that in the package insert, then a more extensive method validation (not simply a verification) must occur and the test is then considered "off-label use" or a laboratory developed test (LDT), depending on the extent of deviation from approved protocols. This makes the user legally responsible for complete validation of the assay5. This article focuses on the process for implementing an FDA-cleared assay in your laboratory. The parameters of a commercial test that a laboratory must document in their own performance of the test are accuracy (analytical sensitivity and specificity), precision (reproducibility), reference range (for qualitative assays), and reportable range (for quantitative assays).

Flu Season 2011-2012

Friday, December 23, 2016 Published in On-Demand Winter 2012 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Laboratories performing tests for respiratory viruses can attest to the changing epidemiology of this formerly reliable season. For many years, like clockwork, new influenza strains, having mixed and matched up genetic material from pigs, birds, and humans, originated in Southeast Asia. In the past, the first influenza cases of the year appeared in Australia in June, peaked in late July, and petered out around December.1

From the Editor

Friday, December 23, 2016 Published in On-Demand Summer 2011 Written by David Persing M.D., Ph.D., Chief Medical and Technology Officer, Cepheid

This edition of the Newsletter focuses on the challenges of detecting methicillin-susceptible Staphylococcus aureus (SA) and methicillin-resistant S. aureus (MRSA) by using molecular assays. Several companies today offer a range of products for SA and MRSA, so users understandably may be confused as to which assay to use. Dr. Ellen Jo Baron explains some of the obstacles that test manufacturers face when developing assays and keeping them current as this elusive microbe changes under our noses (pun unintended) in ways that could affect test performance. Her article highlights the differences between the two Cepheid nasal surveillance assays and presents our opinion on the optimal use of each assay. In fact, there is even a quiz for you to assess your understanding.

Decisions, decisions: Which Staphylococcus Surveillance Assay Should You Use?

Friday, December 23, 2016 Published in On-Demand Summer 2011 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

QUIZ: How should this result be reported to your clinicians?

No two healthcare systems are alike, nor are the patient-care needs of your clinicians, so Cepheid offers choices for detecting both staphylococcal infections and staphylococcal colonization. In fact, there are more FDA-cleared Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) assays available for GeneXpert® System than for any other diagnostic platform. Although it is more obvious in which situations the Xpert® MRSA/SA Blood Culture Assay or the skin and soft tissue infection (SSTI) assay (Xpert® MRSA/SA SSTI) should be used, the choice between the two nasal surveillance assays is less clear. The performance characteristics of each assay are slightly different, so each assay has a unique place in an overall surveillance program. The original surveillance tool, the Xpert® MRSA assay, is the most utilized molecular MRSA surveillance method in the U.S. This assay detects only a single staphylococcal target, which is the junction where the staphylococcal cassette chromosome mec (SCCmec) [i.e., the genetic element that contains the mecA gene, which confers resistance to the extended-spectrum penicillins and cephalosporins], integrates into the S. aureus chromosome. Published data suggest that decades ago, a wild-type S. aureus strain [i.e., one susceptible to oxacillin, methicillin, and other extended-spectrum beta-lactam agents] acquired a large DNA segment containing mecA and the genes that enable it to integrate into another DNA molecule, from another organism (probably Staphylococcus sciuri). Once the DNA cassette was integrated into the chromosome, the mecA gene proved to be fully functional and the newly resistant organism began to spread.1 The integration site of the cassette into the staphylococcal genome is located in the middle of open reading frame X (orfX), a genetic region of unknown function, but one that is unique to S. aureus strains. SCCmec, with its internal mecA gene, inserts into the orfX and in so doing, splits orfX into two parts. The Xpert MRSA surveillance assay targets one set of primers that bind to orfX in the wild type staphylococcal chromosome and additional primers that bind to the outer edge of the newly incorporated SCCmec element, bridging the integrated DNA and chromosomal DNA, insuring that the SCCmec element is present in an S. aureus strain and not some other staphylococcal species. This target is called the SCCmec/orfX junction region (Figure 1).

Prize-Winning Case Report

Friday, December 23, 2016 Published in On-Demand Summer 2011 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

From Dr. Anne Dubouix, Director of Laboratoire de Microbiologie, Clinique de L'Union, Toulouse, France. (Figure 1)

A 44-year-old male presented to the Emergency Department of our institution on November 29, 2009 with inability to ambulate and pain in the right knee. On examination, the patient had a large knee effusion with significant erythema.

The patient reported that his problems with his knee had started nearly two weeks before he finally presented to the hospital. He also stated that because he had been previously diagnosed with sinusitis, he had been taking pristinamycin (similar to quinupristin/dalfopristin, trade name Synercid®) for the previous 8 days as prescribed by his general practitioner.

Laboratory tests revealed a slightly above-normal leukocyte count (11,300/mm3) mainly due to an increase in polymorphonuclear leukocytes (9,800/mm3) and an elevated C-reactive protein (230 mg/l).

Summaries of selected abstracts presented in Europe recently on the use of GeneXpert® MRSA/SA SSTI

Friday, December 23, 2016 Published in On-Demand Summer 2011 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

From the Editor

Friday, December 23, 2016 Published in On-Demand Spring 2011 Written by David Persing M.D., Ph.D., Chief Medical and Technology Officer, Cepheid

I am privileged at Cepheid to work with the some of the best minds in the diagnostics business. In this month's issue of the On-Demand newsletter, Dr. Ellen Jo Baron highlights the role of decentralized molecular testing in TB diagnostics, with a special focus on the nearly impossible challenge of diagnosing pediatric TB. This is a truly moving story which begs the question: Is there not a better solution? Cepheid is committed to developing less invasive diagnostic tools for TB in kids and in all patients with HIV where current diagnostic options are, simply put, woefully suboptimal. I am optimistic that Ellen Jo and her colleagues inside and outside the company will come up with a better way.