Oral Exams

Saliva could provide an alternative for some diagnostic tests

When I was a kid, my best friend Ben and I used to compete at great lengths to make each other laugh. One time, I made Ben laugh so hard that he actually spit, and some of his saliva landed on my arm. I thought that was gross and recoiled in horror, but Ben just laughed even harder. “It’s okay, silly,” he said. “Spit is just water.”

BACTERIAL SIGNAL. Saliva containing unusually large amounts of Streptococcus mitis, which form in chains as seen here, and two other bacterial species can signal the beginnings of oral cancer. A. Tanner

If only he had known what spit scientists know now, he wouldn’t have been so flippant. Besides the water, saliva is swimming with bacteria, viruses, DNA, RNA, proteins, fatty acids, and hundreds of other molecules. Medical scientists are taking advantage of these components to develop tests for myriad health problems. Saliva tests for cancer, diabetes, infections, and the tendency to develop cavities are currently in the works.

Many researchers say that these tests have the potential to transform much of medical diagnostics into a more convenient, less expensive, and less painful exercise, giving spit a long overdue public relations boost.

Spitting’s image

The idea of using saliva as a test isn’t new. According to David Wong, a biologist at University of California, Los Angeles (UCLA), people in ancient times used spit as a primitive lie detector. After asking their suspect a question, early interrogators would pass the individual a handful of rice. If that person had trouble swallowing it, the probable conclusion was that he or she had lied. A dry mouth, the authorities reasoned, had resulted from the stress of being untruthful.

Not until the 20th century did saliva testing begin to find a place in Western medicine. Wong blames this slow progress on spit’s foul reputation. Although some cultures give oral fluid a positive spin—in traditional Greek weddings, for example, people spit on the bride’s gown for good luck—for most people, saliva has negative connotations.

“Saliva is typically viewed as a dirty fluid,” says Wong. “People say, ‘I spit on you, I spit on your grave.'”

Around 50 years ago, researchers’ stance on saliva started to change. Because the 1.5 liters of saliva that a person produces each day come from blood filtering through the salivary glands, scientists discovered that the bodily fluids share most of their substances—though not necessarily in similar concentrations. What doesn’t make it through the filter, and instead remains in the blood, are the blood’s various oxygen-carrying and immune-related cells. “Saliva mirrors our blood because it’s taken directly from it,” notes Wong.

Medical researchers and caregivers have long assumed that people would more gladly spit in a cup for medical tests than submit their arms for needlesticks. Thus, over the past few decades, scientists have developed saliva tests for a range of purposes, among them measuring hormone and drug concentrations, checking identity and paternity, and screening for antibodies against HIV, the virus that causes AIDS.

But, scientists ran into obstacles while trying to develop saliva tests for many diseases, such as diabetes and some types of cancer, that can be detected in blood. That’s because the saliva concentrations of molecules that are the targets of these tests are 0.1 percent to 1 percent of those in blood, says Daniel Malamud of New York University. “What that means is you need more-sensitive assays” to detect the molecules’ presence, he says. With the availability of new, ultrasensitive molecular-detection technologies, the pipeline for new medical saliva tests is now beginning to fill, says Malamud.

Oral fixation

One malady for which saliva testing is quickly proving worthy, says Wong, is oral cancer. The disease is newly diagnosed in more than 350,000 people worldwide each year and can be disfiguring or deadly if not diagnosed early. Although many dentists give their patients yearly exams for oral cancer, it’s often difficult to detect before it progresses to a dangerous stage.

In the Dec. 15, 2004 Clinical Cancer Research, Wong and his colleagues detailed a new method for diagnosing oral cancer using messenger RNA (mRNA) in saliva. The molecule, which serves as a cell’s template for translating the information encoded in genes into proteins, is present in low concentrations in saliva.

When a person is sick, “mRNA is a reflection of the total response of an individual’s disease process and the body’s reaction to it,” says Wong. With that in mind, Wong and his colleagues wondered whether people who develop oral cancer share similar changes in their mRNA.

To investigate, the team recruited 64 people. Half of them had recently been diagnosed with oral cancer and the other half had healthy mouths. After the volunteers provided saliva samples, the researchers examined the approximately 3,000 different mRNA segments present, looking for distinct differences between the two groups of volunteers. In the end, the team identified four pieces of mRNA that showed a characteristic pattern in people with oral cancer. The profile distinguished people with oral cancer from others with an accuracy of more than 90 percent.

Since the mRNAs in saliva are a reflection of the mRNAs in a person’s blood, Wong says that a saliva test could also detect other types of cancer or different diseases occurring elsewhere in the body. He and his colleagues are working to identify the signature mRNA markers in saliva for breast cancer, diabetes, and other health problems.

A group led by Donna Mager, a microbiologist at the Forsyth Institute in Boston, recently announced a different strategy for detecting oral cancer: examining signature populations of bacteria in saliva.

According to Mager, the balance among the 700-or-so bacterial species that live in a healthy person’s mouth is relatively stable. But when someone gets sick, conditions in that person’s mouth can change in ways that alter the bacterial demographics. Mager and her colleagues analyzed 90 volunteers’ saliva to determine whether certain bacterial species consistently prospered or suffered in people with oral cancer. Half of the volunteers had oral cancer; the others were free of the disease.

Although several bacterial species’ had noticeably different population numbers in the two groups of volunteers, the researchers found unusually large populations of three particular species—Prevotella melaninogenica, Capnocytophaga gingivalis, and Streptococcus mitis—in the people with oral cancer. Rather than causing oral cancer, the three signature bacterial species are probably just “innocent bystanders” that reflect a person’s altered body chemistry, Mager says.

Nonetheless, looking for the presence of this bacterial signature in volunteers accurately identified 80 percent of volunteers with oral cancer. Mager adds that incorporating more species into a test for oral cancer could push its accuracy higher.

The research team has recently started looking for other oral bacteria that could signal the beginnings of diabetes.

Infection connection

According to New York University’s Malamud, oral microbes can also indicate viral and bacterial infections elsewhere in the body. The device that he and his colleagues are developing could quickly detect specific pathogens, such as those that cause respiratory infections or food poisoning, without the need for time-consuming culture techniques.

At the heart of the group’s approach is a cassette about the size of a pack of cigarettes that houses a veritable biochemistry-lab. Each cassette is tailored to look for signs of specific bacteria or viruses.

Inside are tiny channels that shuttle droplets of saliva, squeezed from a small, saliva-soaked sampling sponge, to four analysis centers. One of these centers detects human antibodies to a particular bacterium or virus. Another detects proteins that the pathogen carries on its surface. The other two centers amplify the tiny amounts of a pathogen’s DNA and RNA in the sample and probe for genes that can signal the presence of particular bacterial or viral species.

In each center, target biomolecules in the saliva bind to what is effectively a selective lure. When that happens, the resulting complexes migrate to a capture zone via microfluidic pathways etched into the cartridge. Because the lures are labeled with particles that show up as different colors when scanned with a laser, one scan of the capture zones at the end of the analysis should enable doctors to identify pathogens in a patient from a small sample of saliva.

The whole process takes less than 1 hour. Many bacterial or viral tests currently in use take hours or days, notes Malamud. He and his team recently tested a prototype of their system on saliva spiked with HIV and Bacillus cereus, a bacterium related to the one that causes anthrax. In that trial, their system accurately detected both types of pathogens using a single cassette, says Malamud.

Because of these encouraging results, the researchers are working with the Bethlehem, Pa.–based OraSure Technologies, a firm that markets saliva-based drug tests. One idea in the works is a cassette for simultaneously diagnosing several childhood respiratory infections. If doctors could determine the source of a child’s infection within minutes at a clinic, Malamud says, they could avoid prescribing antibiotics unnecessarily for infections caused by viruses, which aren’t affected by the drugs.

Cavity fighter

For his part in the saliva-test movement, developmental biologist Paul Denny of the University of Southern California’s School of Dentistry is working to predict lifelong risk of developing cavities.

For more than a decade, Denny and his colleagues have been studying salivary glycoproteins. These substances float in everyone’s mouth, where they coat and lubricate teeth, protecting them from the daily grind of eating and drinking. Each glycoprotein molecule sports sugar chains that jut out as bristles on a bottlebrush do. The types and combinations of the sugars are genetically determined.

In earlier work, Denny’s team found that the bacteria that cause cavities selectively latch on to some types of glycoprotein sugar chains and are repelled by others. The researchers suspected that the chains, as well as oral hygiene, might determine whether someone ends up with a mouthful of fillings or sails through life cavityfree.

They tested their idea by analyzing the saliva of 29 children, ages 7 to 10, and of 20 adults, ages 24 to 34. To test for a sugar chain, the researchers put a dot of each volunteer’s saliva onto a piece of nitrocellulose paper, then bathed the paper with a marker chemical that’s attracted to the given sugar chain. It took several tests to reveal what combination of sugar chains was present on the glycoproteins in each volunteer’s saliva.

With both age groups, the researchers found that certain combinations of sugar chains correlated with a person’s cavity history, give or take one cavity. People with a higher ratio of bacteria-attracting sugar chains to bacteria-repelling chains had more cavities than people with a lower ratio did.

The inherent value of this test is not in retelling someone’s known cavity history, but in predicting how that person’s dental health is likely to unfold. Since the particular lineup of glycoproteins in a person’s saliva stays the same throughout a lifetime, Denny says that testing a child’s sugar-chain combination would give a dentist a sense of whether a young patient is at a high or low risk of developing cavities. This knowledge could prompt the dentist to take extra precautions—such as applying dental sealants or fluoride treatments—before a child gets his or her first cavity.

Denny’s team is working to develop a marketable test for oral glycoproteins.

Spit decision

Although many scientists are excited about future saliva tests for cancer, infections, and cavities, UCLA’s Wong warns that, for diagnosing many diseases, the strategy will never replace blood tests or other frequently used tools. Some molecules and microbes simply aren’t present in saliva. Moreover, some saliva tests have proved to be less sensitive than the tried-and-true diagnostic techniques that use other bodily fluids are.

Even with these caveats, Wong envisions that the progress of his research team and other groups will eventually elevate spit to a starring role in medicine. At first, he says, doctors and dentists might use the tests for quick diagnoses that often will require confirmation by conventional examinations or biopsies. In time, he predicts, the tests will be good enough to become stand-alone, diagnostic procedures of choice.

Says Wong, “That’s when the proper respect of saliva will come.”

More Stories from Science News on Health & Medicine