Forward march on commercial NAAT for M. genitalium

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.

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.”

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.”

M. genitalium is often asymptomatic in women, so they don’t know they have it and they don’t get treated, Dr. Manhart notes. But the infection is associated with upper reproductive tract symptoms and sequelae like pelvic pain, infertility, and adverse pregnancy outcomes. “The prospective studies and randomized trials that can show that it causes PID haven’t been done yet.”

Unlike chlamydia, M. genitalium is not reportable, so there is less data. “With chlamydia, we have surveillance data we can track over time,” she says, “and we’re not able to do that with M. genitalium. That said, in the general population, the prevalence of M. genitalium is one to three percent, a little less than chlamydia,” at four percent, but more than twice as common as gonorrhea.

In high-risk populations, the prevalence of M. genitalium can be high, she says, comparing the organism with HIV. “With HIV, people are virtually at an infinitesimally small risk of HIV in a low-risk population, but in higher-risk populations like highly sexually active people with a high rate of partner change, the prevalence can be high—in the case of M. genitalium, as high as 16 to 20 percent, which in some recent studies would be higher than the prevalence of chlamydia.”

To Dr. Manhart, that finding suggests the prevalence may be increasing because the effectiveness of treatment has diminished due to antibiotic resistance. In fact, in an important way, M. genitalium is more like gonorrhea than chlamydia: “Gonorrhea develops resistance to almost every antibiotic you treat it with, and that’s what we’re seeing with M. genitalium.”

The M. genitalium bacterium lacks a cell wall, and because a number of antibiotics work by damaging the cell wall of bacteria (penicillin, β-lactams, glycopeptides), that entire class of antibiotics is not going to work, Dr. Manhart says. “But the greater concern with M. genitalium is that it has a fairly high mutation rate. People get infected with an entire population of organisms and some have a variety of different mutations. And there are single point mutations that confer almost complete resistance to one of the primary antibiotics that is used as syndromic treatment for reproductive tract infections: azithromycin. That is where we are seeing huge emergence of high levels of resistance—in the U.S., probably on the order of 50 percent or greater, depending on your area.”

Dealing with this development has required creativity, Dr. Manhart says. “Some companies have included detection of mutations for the macrolide class of antibiotics in their diagnostic assays. That tells you whether the infection would be susceptible or resistant to azithromycin.” If resistance mutations are detected, the provider can prescribe the more potent broad-spectrum drug moxifloxacin, which is the recommended second-line therapy. If no resistance mutations are detected, the provider can still use azithromycin. M. genitalium has already begun to show resistance to moxifloxacin, she says, so less frequent use will slow that process.

In the U.S., Dr. Geisler notes, the three main classes of antibiotics used to treat M. genitalium are tetracyclines, macrolides, and quinolones. His recent retrospective studies at the University of Alabama looked at testing of HIV-infected men who have had sex with men and testing of a heterosexual population enrolled in a sexually transmitted disease clinic in Birmingham (Dionne-Odom J, et al. Clin Infect Dis. 2018;66[5]:796–798). He found that more than 70 percent of infected men had M. genitalium strains with evidence for macrolide resistance and more than 20 percent had strains with evidence for both quinolone and macrolide resistance. “In the tetracycline class, doxycycline is not a very good antibiotic; it cures only 30 to 40 percent of infections. In the macrolide class, azithromycin historically was a good antibiotic, but now cure rates for M. genitalium urethritis are below 50 percent.”

In sum, he says, “We found that the resistance, even in Alabama, is high enough at this point that we have some patients we might not be able to effectively treat with the antibiotics we have available in the U.S.” Newer antibiotics available in Europe and elsewhere outside the U.S. appear to be effective against M. genitalium. Pristinamycin, made in France, is one example. “But you can’t get it here unless you go through the FDA to get it imported and have special circumstances.”

Dr. Geisler’s findings are concerning, Dr. Manhart says. “Macrolide resistance correlates very well with treatment failures. We know when people have detected macrolide resistance, they probably are not going to be cured if they’re given azithromycin. For quinolone resistance, the mutations have been less consistently correlated with treatment failure.”

A couple of studies already in the literature have demonstrated this inconsistent correlation of quinolone resistance-associated mutations and treatment failure, and a forthcoming UW study has also shown this, she says. “So clearly we don’t know the significance of those mutations for quinolone resistance. In some cases, treatment failure results—but not always. We have a little more work to do to understand which mutation—and maybe it’s more than one—and how the mutations actually result in failure of moxifloxacin to cure the infection.”

“I would say we’re at the tip of the iceberg stage, meaning there will be much more demand for M. genitalium testing in the future, and depending on where you are, pairing it with some type of resistance detection, at least of macrolide resistance, will be important.” In Seattle, she says, “Our level of resistance is already so high that it would not be of benefit to detect macrolide resistance, because most already have it. We would just go immediately to treating them with another antibiotic. But in other places where resistance is lower, it would be important to be able to do both: detect the organism and detect whether there was macrolide resistance.”

Specimen collection with M. genitalium, as with any STI, has its complexities, Dr. Manhart notes. “I think most of the forthcoming M. genitalium assays can use urine specimens, and that’s great, because those are easy specimens to obtain, particularly for men who don’t want to have a urethral swab. Self-obtained vaginal swabs, where a woman collects the specimen herself, are the best specimen for women.” The assays are versatile: “They perform well on urine, vaginal swabs, cervical swabs, or urethral swabs.”

Less work has been done to validate oropharyngeal sampling and rectal sampling for M. genitalium, Dr. Manhart says. “Those are sites that are frequently sampled when you are looking for STI pathogens like gonorrhea or chlamydia. There’s been less work done to validate them on M. genitalium, but I’m certain that’s on the horizon.”

How much female reproductive sequelae does M. genitalium cause? That is the million-dollar question, Dr. Manhart says. “A number of public health authorities feel we don’t know how important M. genitalium is until we answer that question.” The thinking is, “If it doesn’t cause infertility and adverse reproductive health outcomes, then maybe it’s not such a big problem.”

CDC guidelines set the standard of care, and the CDC does not recommend screening now at the national level because there is not yet consensus about M. genitalium causing reproductive complications, so that often means an insurance company might not cover costs for M. genitalium testing, Dr. Geisler notes. “But even the CDC says certain individuals do warrant diagnostic testing if there is access to the test, particularly men with recurrent or persisting urethritis. If you have access to a commercial lab with a test that is validated, CDC does recommend that you think about doing diagnostic testing in that setting.”

To resolve some of the open issues, Dr. Geisler says, sometimes only retrospective studies of data collected for routine care are practical, because finding money to fund a new prospective study in which people are enrolled is difficult. Once a commercial test is approved and people start using the assay more, better research can be done. “Right now we don’t have enough testing going on, so we don’t have access to a lot of the data we need.”

Even though there is still much to learn about the epidemiology of M. genitalium, Dr. Geisler says, “we’re already losing time with resistance, and we need to move past just focusing on epidemiology studies and need to start investing more in testing and treatment strategies.”

“We may be entering an era where we won’t have drugs to treat this infection. We need a new drug in the U.S. to treat M. genitalium because of the resistance. That means either clinical trials of drugs already being used outside the U.S., which takes time and money, or having discussions with the FDA about a process to bring drugs already studied and used outside the U.S. into the U.S.”

The second priority, he says, is research to start development of a vaccine. “You can’t develop a vaccine if you don’t understand how the body fights the infection, and here we really don’t understand that or what M. genitalium proteins we might want to target for vaccine development. But unfortunately, so far there is very little research contributing to vaccine development.”

Dr. Manhart believes it could be 12 to 18 months before the FDA approves one of the two M. genitalium tests pending at the agency. Dr. Geisler, too, estimates approval sometime before the end of 2019. But in Europe, all kinds of M. genitalium tests are approved, Dr. Manhart notes. The Hologic and SpeeDx tests have a CE mark, along with a number of others. Testing and treatment guidelines for M. genitalium have been released in Europe and Australia, most recently by the British Association for Sexual Health and HIV, which published guidelines in July. “So other places in the world, for the last couple of years, have had much better access to M. genitalium testing than we do,” Dr. Manhart says.

The U.S. won’t be far behind, Roche’s Dr. McGowin says. “At some point in the near future, M. genitalium tests will be widely available that are FDA approved, and ideally there will be guidance from the CDC on how often we should screen for the pathogen.”

Anne Paxton is a writer and attorney in Seattle.