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Forward march on commercial NAAT for M. genitalium

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Anne Paxton

September 2018—About five years ago, when William M. Geisler, MD, MPH, was still focusing his research at the University of Alabama at Birmingham on chlamydia, Mycoplasma genitalium carried a lower profile as a cause of sexually transmitted infection. M. genitalium is often asymptomatic, and although there was early limited evidence linking the bacteria to reproductive complications, it was insufficient to warrant widespread testing for M. genitalium in asymptomatic persons. Diagnostic nucleic acid amplification tests were available in a limited number of laboratories but were not FDA approved and thus could not be marketed.

But M. genitalium is a more prevalent STI than gonorrhea, and Dr. Geisler worried that M. genitalium could become a serious public health hazard. “As I continued to follow M. genitalium clinically, I felt it was clear this was a pathogen that was definitely causing clinical syndromes—and it was evolving. This bacterium was rapidly developing resistance to some of the only drugs we had in this country to treat it, so it would be a very difficult STI to manage in the future. And I felt compelled to get involved in the research,” says Dr. Geisler, a professor of medicine and epidemiology and clinical associate director of the Medical Scientist Training Program at UAB.

‘Gonorrhea develops resistance to almost every antibiotic you treat it with, and that’s what we’re seeing with M. genitalium.’ —Lisa Manhart, PhD, MPH

He has now led several studies of diagnosis and treatment of M. genitalium. But, as Dr. Geisler emphasized in an Association for Molecular Pathology webinar in February, “The lack of a commercially available, FDA-approved M. genitalium test for the organism is a key limitation in managing and preventing the infection.” With diagnostics companies now preparing applications for their M. genitalium tests for FDA approval, and additional companies perhaps to follow soon, the picture is likely to change.

M. genitalium has many parallels with chlamydia, “which is why I felt I could slide into this field,” he says. But the course of M. genitalium test development has been somewhat different. When chlamydia testing came on the scene, people had already accepted that the STI was a problem. “With M. genitalium, there is building evidence that it causes upper genital tract infection and complications like pelvic inflammatory disease and infertility. But there isn’t yet the kind of stronger research evidence of complications that we had with chlamydia.”

More significant is the difference in resistance development. “Chlamydia, which we’ve been treating clinically with azithromycin and doxycycline for over 30 years now, has never developed full resistance to those antibiotics,” while in 10 years azithromycin has gone from being a good treatment for M. genitalium to being a poor treatment because of resistance, Dr. Geisler says. The cure rate for M. genitalium using moxifloxacin has declined from almost 100 percent to, according to some studies, less than 90 percent.

“The diagnostic companies had been trying to figure out how important it is and whether they want to invest money in developing a test and bringing it to market. Now with research evidence demonstrating how common the infection is and how difficult the infection is becoming to treat, the companies are moving forward with securing approvals for their tests. We’ve certainly seen a much bigger push by researchers, particularly in the pharmaceutical industry, to start studying different tests,” he says.

A fair number of U.S. labs have an in-house PCR-type assay for M. genitalium, in some cases a multiplex assay that may test a whole panel of different pathogens, including those causing chlamydia, gonorrhea, and genital herpes. “The problem with all of these is: How good are their tests? You don’t know,” Dr. Geisler says.

M. genitalium has unique traits that make nucleic acid testing desirable, says Chris L. McGowin, PhD, scientific affairs manager for Roche Diagnostics, which has an M. genitalium test in development. “The bacterium is morphologically too small to be detected in Gram stains of urogenital smears, it requires weeks to months for culture, and serological testing has proven complicated due to cross-reactivity with Mycoplasma pneumoniae.”

Although M. genitalium has been known since 1980 and has been a concern, M. genitalium testing in the U.S. is still sparse, he explains, even with several major reference labs like LabCorp and Quest offering laboratory-developed tests, or LDTs.

“It doesn’t serve the patients well to wait for a company-developed test that’s FDA approved and commercially available,” Dr. McGowin says, “so LDTs are really an interim solution in the U.S.”

Roche is in the process of applying for FDA approval of its M. genitalium test, but because the assay is still in development, few details are available publicly except that it is a NAAT, says Dr. McGowin. NAATs are the only tests used clinically today for M. genitalium, he adds, because they have a good turnaround time, are fairly cost-effective, and culture and serological tests are not clinically practical.

There is no market for a quantitative test for M. genitalium. “From a diagnostic perspective there’s not a lot of difference between M. genitalium and chlamydia or gonorrhea. Based on our current understanding, we’re interested in whether the organism is there or not, not how much or how little. If it’s there, it’s not supposed to be, and it warrants treatment.”

Dr. McGowin

In terms of clinical utility, the most important parameters for M. genitalium NAATs, or for any STI test, says Dr. McGowin, are analytical sensitivity and specificity. In general, STIs, and particularly M. genitalium, don’t colonize the reproductive tract at a very high organism burden, so sensitivity is important for detection. “But specificity, arguably, is even more important, because if you think about the lower reproductive tract, particularly in women, there is a multitude of other colonizing organisms. So the capacity of the NAAT to specifically detect M. genitalium, gonorrhea, or chlamydia is extremely important.”

For example, aside from M. genitalium, “There are several other mycoplasma species that colonize the reproductive tract normally that are not disease-causing. Mycoplasma are kind of a unique group of organisms; the vast majority have no role in the disease that we know of at all.” Manufacturers like Roche, and certainly every other manufacturer, would want to do their due diligence in assessing specificity to a high level because of the potential for cross-reactivity, he says.

Roche’s M. genitalium test is coupled with a Trichomonas vaginalis test as a duplex that is already offered in Europe and labeled TV/MG. “The tests can be run separately, but much like chlamydia and gonorrhea, the thought is they will be run together,” Dr. McGowin says.

Two other companies now in the process of seeking FDA approval for an M. genitalium assay are Hologic and the Australian company SpeeDx, says Lisa Manhart, PhD, MPH, professor of epidemiology at the University of Washington School of Public Health. The Hologic test can be purchased now as long as purchasers agree to do their own in-house validation to use it for patient care. “The challenge there,” she says, “is that there has to be enough demand to make it worthwhile to go through the validation process and purchase the test.” At UW, “Hologic’s is the test we use in our research studies and we have validated it in our research labs, so we do use the results for clinical care.”

What makes FDA approval the holy grail, Dr. Manhart says, is not only that it authorizes companies to market the test but also that it is required for public health agencies to develop guidelines for testing and treatment. “The test manufacturers have typically felt the need to wait until they know there is going to be a big enough market for them to make it worthwhile to undergo the FDA approval process, because it’s very expensive.”

M. genitalium and chlamydia share many traits, Dr. Manhart says. “Both infect the reproductive tract of men and women, they have the same clinical manifestations, both are often asymptomatic, and both have a similar amount of evidence in the literature tying them to male reproductive tract syndromes like urethritis.”

But unlike chlamydia, the M. genitalium bacterium is extremely difficult—almost impossible—to culture, Dr. Manhart says. “To my knowledge, there are only three labs in the world able to do it,” UW among them. “Even though the procedures are published, M. genitalium is just a fickle, finicky organism, and it has to grow in Vero cells. Only the ATCC strain can grow on a plate.”

In this respect, M. genitalium has similarities with Treponema pallidum, the spirochete bacterium that causes syphilis, Dr. Manhart notes. T. pallidum cannot be cultured; it has to be grown in a rabbit. “We’re learning, with newer sequencing technologies that are used to study the microbiome, that there are lots of bacteria out there that we don’t know about because we can’t culture them yet. I think M. genitalium is at the forefront of organisms that are very difficult to culture. But it’s certainly not the only one.”

With serological tests, which look for antibodies to M. genitalium, the number of false-positives is very high. In addition, as Dr. Geisler notes, antibody tests don’t distinguish past exposure from current infection. “We really don’t have any good serological tests,” Dr. Manhart says, “not even in the lab. And that continues to be a challenge for M. genitalium.”

The NAATs are better. “Although no test is perfect, the two diagnostic assays moving forward for FDA approval have very low rates of false-positives and false-negatives,” she says. “When people develop NAATs, they usually do a pretty rigorous evaluation to figure out the likelihood of false-positives and false-negatives. When you can’t culture a bug, as you can’t do with M. genitalium, they use another procedure called ‘patient infected standard.’ That involves NAAT testing of several different types of samples from a given patient. The gold standard is defined as a positive result on any two of the three samples. If you have only one out of three, then the gold standard is to say the result is a negative.”

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