Friday, December 23, 2016 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Next-Generation Testing for CT/NG

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.

Xpert CT/NG: the Test
The development of the Xpert® CT/NG test has taken longer than anticipated. Although most of the Xpert assays have the inherent advantages of being moderately complex (can be performed in almost any clinic or laboratory setting) and a turnaround time fast enough to potentially deliver results during the patient's initial clinic visit, Cepheid chose not to compromise performance for expediency. Many of the problems associated with previous "rapid" tests (compared to culture), as elegantly presented by Dr. Julius Schachter in his 1986 paper "Rapid diagnosis of sexually transmitted diseases: speed has a price," have been solved.1

The clinical trials of the first version of the CT/NG product demonstrated less than desired sensitivity, and rather than release it as it was, even in response to many calls from public health officials who felt that a slightly lower sensitivity was an acceptable alternative to losing as many as 30% of patients to follow-up, Cepheid stopped the trials, abandoned the initial cartridge, and Development Scientist Fred Weir pulled the assay back into the research and development laboratory for a makeover. The result exemplifies Cepheid's approach to product development: the assay should not only be the easiest to perform NAAT with the fastest time to result, but it should be equal to or better than the best-in-class first generation tests. This test is the first test likely to satisfy most of the desired characteristics of the hoped-for point of care test requested by an online survey of practitioners: high sensitivity and specificity, quick turnaround time, and low cost.2 The new Xpert CT/NG will be the first NAAT that can be used as a STAT, random access test with turnaround time expected to be just under 90 minutes.

Novel Genetic Targets
The assay design has benefited from using sophisticated electronic subtraction design strategies, benefiting from the problems of earlier NAATs. Instead of choosing genetic targets that are easily shared among other species of Neisseria, the scientists at Cepheid chose highly conserved and species-specific targets. In fact, there are two of them, to drastically reduce the possibility of a false positive result for NG. High concentrations of Neisseria cinerea, N. meningitidis, and many related Neisseria, Moraxella, and related strains have been tested with no incorrect results.

This same strategy was used for C. trachomatis; rather than target a plasmid sequence, which despite delivering better sensitivity because of its multiple copy numbers, is potentially (and actually) labile, Cepheid chose chromosomal DNA targets. For C. trachomatis; more than 15 serotypes have been successfully detected, including the new Swedish strain that is missing the plasmid targeted by several commercial assays. This is particularly important, as the loss of plasmid targets resulting in false negative results in these assays was identified as a root cause in unabated outbreaks of C. trachomatis in Sweden.3

Xpert CT/NG: Molecular Testing for Control Freaks
An expected feature of all GeneXpert assays, and of all next-generation NAAT technologies, is inclusion of a process control in which the control is added to the clinical sample before the test actually starts, and is then carried through the process of concentration, purification, amplification and detection, thus controlling for all aspects of the testing process. An additional first-in-class feature is a sample-adequacy control that ensures that the sample has human cells within it. This will become increasingly important as laboratories and public health authorities move to more self-collected vaginal swab testing algorithms. Each cartridge, run as an independent test, contains all the quality control checks necessary. Other batch-mode NAAT assays require that controls be included with each run, increasing the cost of the assay, and also precluding testing if only small numbers of samples have been received, as the systems are designed for maximum efficiencies when testing a full load. The inclusion of an internal control allows laboratory directors, after their initial validation, to determine the frequency with which an external control is used to control the performance of the assay. Another aspect of using the Xpert test as it was optimized to be used, in the random-access "as-samples-are-received" mode, helps remove the root cause of false-positive results due to contamination carried over from a highly positive sample to a negative. Avoiding batch testing has many advantages.

Way of the Future: Accuracy by Design
The assay incoporates a unique PCR protocol developed by Dr. Russ Higuchi, one of the inventors of real-time PCR. A special cartridge design promotes DNA binding to a fiber matrix in one of the cartridge compartments to maximize detection of CT. Samples in the Cepheid transport medium have room temperature stability for 3 days (female urine), 30 days (male urine), and 60 days (swabs) and even longer if refrigerated. Urine alone (without transport medium) can be tested for up to 6 days if refrigerated and within 24 hours if kept at room temperature, although laboratories will need to validate this approach since only urine in transport medium is covered in the product insert. The limitation of detection for elementary bodies of CT will be from 1-10/mL and around 1 cfu/mL for NG, whether from swabs or urine. A few substances can cause inhibition, including >1% blood and mucin, and >0.2% Vagisil feminine powder, neither of which are likely to be found at such concentrations in clinical samples. In initial testing in a number of laboratories in U.S. and Europe, the Xpert CT/NG has shown <3% indeterminate results on initial testing and 0.4% indeterminate results after repeat testing. A 1 ml sample of GeneXpert transport medium (for female endocervical or self-collected vaginal swabs or urine) is added directly to the cartridge. In more than 1500 patients tested so far, the sensitivity vs. patient infected status ranges from 96-100% and the specificity was >99.6%. The test has been CE marked and is available for distribution in many countries of the world now.

We feel that this GeneXpert test embodies the latest in the evolutionary progression of test technologies for C. trachomatis and N. gonorrhoeae. It employs a highly stable and specific set of targets, ease of sample collection, and minimal manipulation prior to testing (the hallmark of almost all Cepheid GeneXpert assays), a turnaround time fast enough to deliver results while the patient waits and certainly before the patient has time to infect a new partner, and the results matching the best of class for both sensitivity and specificity. In short, this next-generation NAAT technology meets the next generation of molecular diagnosticians, breaking down barriers to testing and maximizing medical impact along the way.

REFERENCES

1. Schachter, J. (1986). "Rapid diagnosis of sexually transmitted diseases – speed has a price." Diagn Microbiol Infect Dis 4(3): 185-9.
2. Hsieh, Y. H., M. T. Hogan, et al. "Perceptions of an ideal point-of-care test for sexually transmitted infections – a qualitative study of focus group discussions with medical providers." PLoS One 5(11): e14144.
3. Reischl, U., E. Straube, and N. Umemo (2009). "The Swedish new variant of Chlamydia trachomatis (n vCT) remains undetected by many European laboratories revealed in the recent PCR/NAT ring trial organized by IN STAND e.V., Germany." Eurosurveillance 14(32):1-4.