The perils of overlooking lesser-known STIs

“FDA clearance is very time-consuming and costly,” notes Lisa Filippone, business manager in women’s health core diagnostics at Hologic. “For us, it’s really about prioritization. The health care burden for T. vaginalis seems to fall on females, so we focused on that population. But we are looking to fill out our pipeline with relevant assays as fast as we can. Unfortunately, development timelines for new assays are inherently long, especially when seeking FDA clearance.”

As a result, there’s an unmet clinical need with respect to the diagnosis of T. vaginalis in males, says Dr. Munson. In the past, molecular tests for trichomoniasis were limited to PCR assays developed in-house and validated on alternative specimen sources. But the availability of commercial tests now means laboratories can adapt them to a different gender or specimen source. “Since the commercial reagents are out there, it’s a little easier for laboratories to modify the test, validate it, and verify it for the means they find desirable.”

Several years ago, when the transcription-mediated amplification assay for T. vaginalis first became available as an analyte-specific reagent, Dr. Munson’s group not only validated it for female specimens but also performed an in-depth validation of the test for male specimens. “We were able to gather enough data to validate that assay, in-house, off-label, for both male urethral testing and male urine testing,” he says.

Now his laboratory offers that test as an option on its clinical testing menu, with appropriate disclaimers about the alternative specimen sources. But Dr. Munson’s research reveals that surprisingly few clinicians and patients take advantage of the male test. “Our data suggest that we test far more females than males,” he says, adding that he doesn’t know whether that’s because of the perceived limited testing options in men or the tendency of men to not seek health care.
There’s an important take-home lesson, Dr. Munson says: “Don’t forget about men. The male market is out there, available to be tested. If a laboratory has the means to validate a test, it should consider doing that.”

A laboratory’s ability to do so, however, depends on its resources, disease prevalence, and the technical acumen of the laboratory director and microbiologists. “If the laboratory is in an area of high STI prevalence, there will be plenty of organism burden to assist in the validation process,” Dr. Munson says.

His research suggests that trichomoniasis is just as prevalent in suburban as in urban areas, contrary to the popular notion that STDs are more common in cities. “T. vaginalis is more of a community- or metropolitan-wide disease. So if a laboratory is in a high STD-prevalence area to begin with, and they’re already testing for gonorrhea and chlamydia, they might consider doing prevalence studies for Trichomonas vaginalis and then determining whether to develop a test for males.”

The popular misconception that testing options are not available for males is beginning to change with greater attention on T. vaginalis in the peer-reviewed literature, on social media sites, and at national meetings and symposia. “One of the STD clinics we serve actually has male patients coming in asking for the Trichomonas test specifically,” Dr. Munson says, encouraged by the recent development.

Still, additional work is needed to educate patients and physicians. Says Dr. Chapin, “The educational focus we had back in the early 1980s for chlamydia and gonorrhea, we’ll probably need to do for Trichomonas as well.”

Awareness of the FDA-cleared molecularDr. Munson predicts that a newly recognized sexually transmitted pathogen, Mycoplasma genitalium, will emerge soon as a candidate for new diagnostic assays.

“The director of the STD clinic that I deal with always tells me about cases of men with nongonococcal urethritis who have negative chlamydia results, negative gonorrhea results, and negative T. vaginalis results. And in the back of his mind, he’s wondering what the role of M. genitalium might be in these men. I’ve heard discussions at national meetings to the same extent.”

That question has formed the basis of years of research in the laboratory of Pat Totten, PhD, a professor of medicine in the Division of Infectious Diseases and an adjunct professor of global health in the Interdisciplinary Program of Pathobiology at the University of Washington. “There have been many hypotheses about what could be causing nonchlamydial nongonococcal urethritis in men. It turns out, 50 percent of urethritis cases are idiopathic. We don’t know what causes them,” Dr. Totten says.

Her research in the early 2000s helped establish M. genitalium as one of the culprits, eventually showing that the organism was present in 22 percent of men with nongonococcal urethritis, coming in a close second to Chlamydia trachomatis, which was present in 30 percent of the men.

But studying the pathogen wasn’t easy. Describing the organism as merely “difficult to grow” would be a vast understatement. Mycoplasma genitalium was first isolated in 1980 by microbiologist Joseph Tully, PhD, from two of 13 men with urethritis. Dr. Tully reportedly spent months coaxing the organism to grow on solid medium. Eventually, two strains were isolated and submitted to ATCC, but few researchers have been able to isolate those strains in the years since this study.

To demonstrate the association between M. genitalium and urethritis, Dr. Totten relied heavily on the work of a Danish microbiologist, Jørgen Jensen, MD, PhD, who developed one of the first PCR assays for M. genitalium and eventually hit upon a method for growing M. genitalium in tissue culture, treating it as if he were growing a virus. “Dr. Jensen found that he could actually isolate clinical strains, which researchers hadn’t been able to do since the first isolation in 1980 and ’81,” Dr. Totten recalls. “He taught my lab how to grow the strains from clinical specimens, and we’re probably one of the few labs that can grow them now. We have collected more than 100 M. genitalium cultures from men with urethritis and may have a corner on the market now.”

From the initial realization that M. genitalium causes urethritis, it didn’t take long for Dr. Totten’s group and her collaborators to uncover links to a number of other conditions. “We had an old sample set collected from women between 1984 and 1986 and used to show the association of chlamydia with cervicitis in women. So we recovered these specimens from our freezers and found that, sure enough, M. genitalium was also independently associated with cervicitis. Subsequently, we and other research groups showed the association of this bacterium with endometritis, pelvic inflammatory disease, preterm birth, and tubal factor infertility in women, demonstrating that M. genitalium has a disease spectrum very similar to chlamydia.” The association of M. genitalium with these serious diseases and their sequelae illustrate the need to identify and treat women infected with M. genitalium.

On another note, Dr. Totten and her collaborators found an association between M. genitalium and cervical shedding of HIV. “People who have STDs are more likely to shed or acquire HIV, probably because they have a disruption of their epithelial barriers, such as ulcers or micro-lesions, allowing HIV to enter the underlying tissue and come into contact with the appropriate cell types that HIV infects.”

Despite the urgency of treating M. genitalium infections, there are no commercial diagnostic assays to screen for this sexually transmitted pathogen in the United States. “The problem is, it takes so much money and time to get FDA approval that nobody has yet pursued it,” Dr. Totten explains.

Hologic is considering a move in that direction. “Mycoplasma genitalium is very difficult to grow in culture, but that actually makes it even a better target for an amplified assay,” Filippone says. “There’s a need for that test on the market, and it’s something we are considering developing commercial assays for.” Studies have indicated that self-obtained vaginal swabs from women and urines obtained from both sexes are the specimens of choice for M. genitalium NAAT, especially in non-clinical settings, Dr. Totten says.

But even if diagnostic assays were available, the question remains: If M. genitalium is detected, what is the best way to treat it? “In our recent trial for treatment of nongonococcal urethritis in men, headed by Lisa Manhart [PhD], azithromycin and doxycycline were only moderately effective for clinical and microbiologic cure of M. genitalium,” Dr. Totten says. Men who failed azithromycin treatment harbored M. genitalium strains that were resistant to this antibiotic and contained azithromycin-associated mutations that could be detected by PCR. “It would be great if this PCR could be used to guide treatment in the future,” she says.

“So many men failed treatment with azithromycin and doxycycline, the antibiotics commonly used to treat urethritis, and M. genitalium is becoming resistant to azithromycin,” says Dr. Totten, who continues to test various antibiotics against this pathogen. “The bottom line is that M. genitalium is becoming increasingly recognized as an STD pathogen with a significance rivaling that of chlamydia and we need FDA approval to detect it, we need to find antibiotics that can treat it, and we need to continue to find new drugs as the organisms get resistant to the currently used antibiotics.”

While herpes simplex virus hardly falls under the realm of under-recognized STIs, clinicians and laboratories may be surprised to learn of new developments, particularly the upswing in genital herpes cases due to HSV 1, rather than HSV 2.

“HSV 1 has been thought for many decades to be the virus that infects above the belt, while HSV 2 is the one that infects below the belt,” says Rhoda Ashley Morrow, PhD, a professor of laboratory medicine at the University of Washington and head of faculty affairs in the Vaccine and ID Division at Fred Hutchinson Cancer Research Center.

While that still holds true for recurrent infections, she says, most of the newly infected—especially women younger than age 25 in the U.S. and men who have sex with men—tend to have HSV 1. “We have been thinking for so long that a serology that was positive for HSV 2 meant genital herpes, and a serology that was positive for only HSV 1 meant probably not herpes, but that’s really an oversimplification and a disservice to patients.”

Recent research highlights the need for clinicians and laboratories to look more seriously at the potential for genital infections among those who are HSV 1, but not HSV 2, positive.

Currently, there are two approaches to diagnosing active herpes infections: viral culture and the far more sensitive nucleic acid amplification testing, such as PCR. Inactive lesions, or negative lesions with a high index of suspicion for herpes, are diagnosed via serology—a much more challenging approach.

“The serology tests are quite good for HSV 2,” Dr. Morrow says. But her work suggests many of the commonly used tests fall short in the detection of HSV 1 antibodies, probably because it takes longer to demonstrate seroconversion in those who have new genital HSV 1 infection than in those infected with genital HSV 2. “Even our lab’s Western blot, which is considered a gold standard, is a little less sensitive for HSV 1 than it is for HSV 2.”

Dr. Morrow worries that the lack of focus on HSV 1 as a cause of genital herpes means existing tests aren’t necessarily optimized for the best combination. “My lab supervisor, who has been in the lab for 25 years or more, and I joke with each other about how the field has to catch up to us, and get us an alternative to Western blot, if we are ever going to be able to retire. We’re very proud of our high level of accuracy and reproducibility, but there aren’t many options outside of our lab. We’re hoping that somebody out there is putting together the right combination of tests to create an accurate algorithm that’s available for everybody.”

As with most diagnostics, the tradeoff between sensitivity and specificity remains an issue with the ELISAs used in serology testing. “The main herpes blood test that has survived on the market for a decade or more seems to have a gray zone, where low positives in some populations tend to be more difficult to confirm by other gold standard tests like Western blot,” Dr. Morrow says. She cites concerns that African populations and even patients in the United States are occasionally given positive results when they do not, in fact, have HSV 2 infections.

Dr. Morrow’s group and others have attempted to broaden that gray zone by raising the cutoff for a positive result. “Instead of setting the cutoff at an index value of 1.1, we set a higher cutoff and call everything up to a 3.5 equivocal. Depending on the population, we’ve found that up to a third of the low positives we test by Western blot do not have HSV 2 antibodies.”

That’s the opposite problem facing HSV 1 antibodies, she notes. “My concern with HSV 1 is that the tests don’t pick it up. We don’t have a good way of telling all the positives the way we do with HSV 2.”

But a definitive diagnosis, however hard to achieve, is key. “Learning that you have a sexually transmitted infection that is not curable, that you can control but you can never get rid of, is a daunting message.”

If clinicians deliver that message, Dr. Morrow says, laboratories should try as hard as possible to ensure that the test behind it is accurate. “That’s the real issue with HSV 2 testing,” she says. “Tests that are financially reasonable and widely available, like an ELISA, may give the wrong answer part of the time. At the very least, we need clinicians to be aware of that possibility, and to counsel their patients, and to be suspicious that the person is at risk of having that infection.”

The other side of the coin, she says, is that half of people in the general population have antibodies to HSV 1. “That’s the really troubling thing. Because you have this high background of most certainly oral HSV 1 antibodies, it’s hard to discern whether a clinical presentation of genital herpes or genital lesions is due to HSV 1,” Dr. Morrow says. “Until we have markers, or much more sensitive methods of testing for the virus itself, there’s going to be a lot of uncertainty around the diagnosis of genital herpes if the person happens to be infected with type 1.”
A statement prepared by the CDC’s Division of STD Prevention says the following: “We need additional evaluation to understand the benefits of testing, including whether routine HSV 2 testing improves health and reduces spread of infection in the population. In addition, these tests can be expensive; false positive test results may occur in some persons with a low likelihood of infection; and the diagnosis may have adverse psychological effects for some people.”
For clinical laboratory managers, that means gathering as much data as possible to reveal how well existing HSV 1 tests can detect new infections with genital herpes.
“There are very limited data in this arena,” Dr. Morrow says. “If you get a positive culture, or a positive PCR for herpes, from the genital tract, it’s very important to type it. If a lab offers a PCR test that only picks up herpes, they might think strongly about adopting a test that discerns between types 1 and 2. That’s especially important now that we’re seeing more HSV 1. We can’t just assume that anything from the genital tract is HSV 2, so this is going to be very important for patient counseling.”
To help establish an algorithm for diagnosing genital HSV 2, the CDC sponsored a large study of eight commercially available assays. “The numbers are being crunched and the data are being analyzed to determine the raw sensitivity and specificity of each of those commercial tests against Western blot, which has never been done before in that large a setting,” Dr. Morrow says.
The question is how to proceed with a positive test result. “It may be possible to use one commercial test on positive samples or low-positive samples from the Focus Diagnostics HerpeSelect HSV 1 test, for example, and to come up with an improved positive predictive value for a combination test.” Dr. Morrow hopes to find a strategy that circumvents the need to send sera for Western blot, which is expensive, cumbersome, and most often done on a research basis.
“It may be possible to put together from this large [CDC-sponsored] study several options, to start with one test and then follow up with a second. That’s my hope anyway.”
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Ann Griswold is a writer in San Francisco.