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Pepsin Test Can Better Detect Aspiration Reflux, Chronic Lung Disease

by David Bronstein •

 

Pepsin may be the best biomarker yet developed for assessing whether reflux and aspiration of stomach contents into the upper airways is an important contributing factor in pediatric lung disease, according to a team of researchers from the Medical College of Wisconsin (MCW). Their study, published in JAMA Otolaryngology-Head & Neck Surgery, found that more than two-thirds of children with a variety of pulmonary disorders tested positive for pepsin in bronchoalveolar lavage fluid (BAL) (2013;139:996-1001). All of the control BAL specimens, obtained from patients with no history of lung disease, tested negative for pepsin, according to co-author Joseph Kerschner, MD, dean and professor of otolaryngology at MCW.

The results show that the pepsin test, a proprietary Western blot assay developed by the team, is a highly accurate method for assessing aspiration-associated extraesophageal reflux disease (AERD) and, moreover, that AERD may be a previously under-recognized cause of pulmonary symptoms in patients with chronic lung problems.

Given the relative inaccuracy of a lipid-laden alveolar macrophage (LLM) assay, a test that is considered one of the current mainstays for evaluating aspiration in these patients and that was assessed in the same study, the results may prove to be practice-changing, Dr. Kerschner told ENTtoday. “Granted, more work needs to be done to show causality between a positive pepsin test, the presence of AERD and chronic respiratory symptoms,” he said. “But once we get there—and we are confident we will—we really feel this is a groundbreaking paper that will have a major impact on how clinicians ultimately diagnose and treat patients with chronic lung disease.”

The study, which was first reported at the 2013 Spring Meeting of the American Society of Pediatric Otolaryngology, included 76 patients (mean age, 6.5 years): 34 who underwent bronchoscopy, 31 who underwent tracheostomy and 11 who served as controls. Patients in the bronchoscopy and tracheostomy groups had a variety of chronic respiratory symptoms, including recurrent wheezing, chronic cough, recurrent pneumonia, regurgitation, suspected aspiration, and shortness of breath. None of the control patients had such conditions; in most cases, they had undergone the diagnostic lung testing prior to elective surgery and the bronchial aspirate was collected through their endotracheal tube.

The researchers identified pepsin-positive BAL fluid specimens in 25 patients who underwent bronchoscopy (74 percent) and 22 patients in the tracheostomy group (71 percent). As noted, all control specimens were negative for pepsin. LLM results, in contrast, were much less helpful; the macrophage biomarker was detected in BAL specimens from 31 patients in the bronchoscopy group (91 percent), 16 patients in the tracheostomy group (52 percent) and perhaps most importantly, in seven control patients (64 percent).

“Clearly, this shows that LLM testing is not a very sensitive method for assessing these patients,” Dr. Kerschner said. “The pepsin assay, in contrast, was highly sensitive and a reliable marker for AERD.”

LLM Results Not a Surprise

Dr. Kerschner added that it should not be too surprising that the LLM test proved so inferior to the Western blot assay for pepsin. “The LLM test is really a measure of generalized inflammation in macrophages; that’s why it’s such a nonspecific, inaccurate test,” he explained, adding that several previous studies have questioned LLM’s accuracy (Pediatr Pulmonol. 1999;27:419-422).

Why, then, does LLM continue to be used? “Until you actually develop a new, superior test for some physiologic process,” he said, “it’s very hard to underscore the shortcomings of the current screening method.”

In terms of next steps, Dr. Kerschner said that his team plans on submitting a grant for a more extensive clinical trial to the National Institutes of Health, which they hope will strengthen the link between pepsin, aspiration and clinically significant chronic lung disease. “But my own feeling is that this test is ready for prime time now,” he said. “In fact, if you are a believer in what came out of this study, it would suggest that pulmonary physicians who routinely order the LLM test in children when they present to the OR for a bronchoscopy as a means of detecting aspiration should probably stop getting the test and order ours.” Dr. Kerschner’s team is exploring ways to make the test commercially available.

In Absence of Causality, Caution Urged

Warren Bishop, MD, professor of pediatrics at the University of Iowa Carver College of Medicine, and director of the division of pediatric gastroenterology at the University of Iowa Hospitals and Clinics in Iowa City, agreed that the Western blot assay for pepsin is very promising. But what is missing in the current study, he said, “is any clinical correlation between the presence of pepsin and the chronic respiratory symptoms in their subjects,” he said. Thus, he added, “It is very hard to definitely say that pepsin is an effective test to document clinically significant extraesophageal reflux disease that contributes to those symptoms.”

There’s also a “chicken-and-egg” factor to consider, Dr. Bishop said. Some chronic respiratory disease “actually can cause reflux and potentially aspiration,” he explained. “Asthma is a good example; negative chest pressure and an increased work of breathing can trigger the reflux of stomach contents into the airways, which could lead to a positive pepsin test.”

Given such a result, “one might conclude that reflux is contributing to the asthma, when in fact the asthma may be causing reflux.” In that scenario, a positive pepsin test may lead clinicians to focus their treatment strategies on reflux rather than the underlying lung disease.

Dr. Bishop added another caveat: It’s possible that the Western blot assay is so sensitive that it is detecting extremely small levels of pepsin in the airways. Thus, one future refinement for this test could be to add more quantitative information. “Could you create, for example, a panel-type test, where a quantitative control is loaded in adjacent ‘lanes’ so that you could see the strength of the band, with different amounts of pepsin shown? That would give you some idea of how much pepsin is being found in the airways relative to the control, and whether that amount is sufficiently higher to denote some connection to the patient’s pulmonary symptoms.”

Dr. Bishop said his own preference for assessing reflux in pediatric patients with chronic lung disease is to have them undergo esophageal pH impedance monitoring. “When patients experience a symptom, they press a button on the recording device, which allows us to correlate those symptoms with the tracing. For us, this gives a better understanding of whether any reflux detected is indeed clinically significant.” But even this test has drawbacks, Dr. Bishop said. For example, the test cannot correlate chronic respiratory symptoms such as vocal hoarseness with individual reflux events, nor are there absolute cutoffs between normal and abnormal studies.

In fact, “all of the standard tests used to detect reflux are imperfect; that’s the problem we are all dealing with,” he said. “So it is important that we explore other methods for assessing reflux and, on that score, this paper is very stimulating and warrants further study.”

More Work Needed

Dr. Kerschner said that Dr. Bishop’s caveats “hit all of the high points” regarding the pepsin test’s current limitations. “We noted in the paper, for example, that our current technique for detecting pepsin in BAL samples can’t be used for quantifying the amount of pepsin present,” he said. “What we essentially showed is that we were able to [develop] a very sensitive identification test for this enzyme. But what does that mean, clinically? The real key will be the next clinical trials that we conduct, not only in children but also in adults, to see if using this test makes a difference in patient outcomes. We think it will.”

Dr. Kerschner stressed, however, that the need for more research should not obscure the importance of his team’s research. “I think we’ve added to the evidence that underdiagnosed AERD—what we call ‘silent aspiration’—is an important contributor to altered or diminished lung function and that you need a reliable test such as the Western blot pepsin assay that can accurately tell you that such a disease process is indeed taking place,” he said. “Well, you truly did not have such an assay until we completed our study.”

Pepsin: Where Does It Come From and Can It Be Treated?

When otolaryngologists consider the damaging effects of excess reflux of stomach contents, acid is usually thought to be the offending agent. Why, then, are researchers now looking at pepsin’s role in this process? The answer, to borrow a phrase from real estate, is location, location, location, according to experts in reflux disease.

Although acid is indeed the culprit in cases of gastroesophageal reflux disease (GERD), a growing body of evidence suggests that pepsin, an enzyme, is the substance that causes the most damage when the reflux extends beyond the upper esophagus and reaches the pharynx, larynx and lungs. Once present in sufficient amounts, studies have shown, pepsin can cause significant damage by adhering to laryngeal cells and breaking down proteins, among other injurious effects (published online November 10, 2011. Int J Otolaryngol. doi:10.1155/2012/646901). Thus, it’s not surprising that pepsin has been linked to serious lung disease, including acute exacerbations of idiopathic pulmonary fibrosis (Eur Respir J. 2012;39:352-358).

As for the theory that pepsin is a relatively unimportant local phenomenon—that is, something produced endogenously in the upper airways—“the literature is pretty clear on this: it shows that pepsin is produced only in the stomach,” Dr. Bishop said. “If you find it in the airways, you know that stomach contents, in some amount, are getting into those airways and potentially causing some damage.”

Assuming the recent work by Kerschner and colleagues linking pepsin to chronic pulmonary disease is confirmed in larger trials, the next question becomes, how should otolaryngologists treat a patient who has a positive pepsin test?

“I think such a result would make us refer patients not to increased acid suppression, but instead to antireflux surgery such as Nissen fundoplication, because remember, pepsin is not an acid—it’s an enzyme and is not affected by acid blocker therapy to any significant degree,” Dr. Bishop said.

Unfortunately, such surgery carries no guarantees of long-term success. “More than half of the patients in the Kerschner study who had undergone anti-reflux surgery actually had detectable levels of pepsin in their tracheas, presumably because those fundoplication wraps were breaking down over time and were not preventing reflux,” Dr. Bishop said. “So this is a definite challenge we all face in managing these patients.”

How about some type of treatment targeted to pepsin itself? That’s a somewhat tricky proposition, said Dr. Kerschner. “There’s really no ‘anti-pepsin’ medication that directly counteracts its effects, but evidence suggests that pepsin tends to be reactivated at lower pH levels, so counteracting those lower pHs with acid suppression therapy may actually have some beneficial effects.”

Dr. Kerschner added, “We are also working in our lab on a more direct approach—that is, treatments that actually do counteract pepsin activation. But we’re quite a ways off from bringing that to fruition.”

Nancy Bauman, MD, a pediatric otolaryngologist at Children’s National Medical Center in Washington, DC, and an ENTtoday editorial board member, noted that regardless of where pepsin originates, “once it gets into the upper airway, the enzyme can do considerable damage.” Thus, the paper by Kerschner and colleagues “really has the potential to be seminal; this could bring about a significant change in how we view the role of extraesophageal reflux in chronic lung disease, how we diagnose the condition and, hopefully, how we treat it.”—DB