The present invention relates to the clinical differentiation and monitoring of gastrointestinal illnesses. More particularly, the present invention relates to a method for aiding in differentiating irritable bowel syndrome from inflammatory bowel disease by determining the level of total endogenous human lactoferrin in clinical specimens, such as feces, mucus and bile, wherein an elevated level of lactoferrin substantially precludes diagnoses of IBS and other noninflammatory etiologies, and a kit usable in such method. The present invention further relates to a method for quantitating the level of total endogenous human lactoferrin in clinical specimens, such as feces, mucus and bile, to monitor gastrointestinal inflammation in persons having inflammatory bowel disease.
Gastrointestinal illnesses are responsible for an extensive loss of life worldwide. For instance, diarrhea is a major cause of morbidity and mortality in developing countries with an estimated one billion cases of diarrheal diseases and five million deaths in children per year. In the United States, eight to twelve million people are treated each year for infectious diarrhea making up 2.5% of total hospitalizations and resulting in 10,000 deaths. Other gastrointestinal illnesses include inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Annual evaluation for these disorders in the United States results in 1 and 3.5 physician visits, respectively. Symptoms of active IBS and those of active IBD are similar and, accordingly, the two diseases often present nearly identically. However, IBD can be a severe, life-threatening condition and thus quick, accurate differential diagnosis is extremely important.
IBD, comprised of both Crohn's Disease (CD) and ulcerative colitis (UC), is characterized by a chronic immune-mediated inflammatory response that results in histologic damage to the intestinal lining. Both CD and UC exhibit large numbers of leukocytes that migrate to the mucosa and into the intestinal lumen. Both diseases oscillate between active (i.e., presence of intestinal inflammation) and inactive (i.e., minimal to no intestinal inflammation) stages of disease activity. Active IBD can include symptoms such as bloody diarrhea, abdominal pain, and fever. The inactive stage has minimal to no intestinal inflammation and lacks severe gastrointestinal illness.
Patients who have active IBD but who exhibit mild signs and symptoms may be difficult to distinguish from patients with active IBS, an intestinal disorder of motility and the intestinal nervous system. Unlike IBD, IBS does not involve intestinal inflammation. In persons with IBS, the intestine appears normal upon endoscopic examination and leukocytes are not present in the mucosa or in fecal specimens. Symptoms can mimic those of IBD and include bloating, diarrhea, constipation, and severe and often debilitating abdominal pain. It is estimated that at least 35 million Americans suffer from IBS.
The similarity in symptoms between IBS and IBD renders rapid diagnosis rather difficult. However, given the potential severity of untreated IBD, differential diagnosis is crucial. The diagnosis of gastrointestinal illnesses, in general, is aided by diagnostic tests such as enzyme-linked immunosorbant assays (ELISAs), latex agglutination and lateral flow immunoassay. These tests are rapid and inexpensive methods for detecting markers in feces for enteric pathogens and inflammation. One marker of particular interest that has been found to be most specific for leukocytes in fecal specimens is lactoferrin. Human lactoferrin is an 80 kilodalton glycoprotein. This iron-binding protein is secreted by most mucosal membranes. It is a major component of the secondary granules found in polymorphonuclear neutrophils (PMNs), a primary component of the acute inflammatory response. Other hematopoietic cells such as monocytes and lymphocytes, do not contain lactoferrin, whereas various bodily secretions contain levels in the mg/mL range. During the process of inflammation, PMNs infiltrate the mucosa lining of the small and large intestine. This increase in the number of activated tissue leukocytes and exudation of plasma from ulcerated mucosa results in an increase in the level of lactoferrin found in feces. The protein is resistant to proteolysis and, as such, it provides a useful non-invasive fecal marker of intestinal inflammation.
Human lactoferrin has been used as a marker for fecal leukocytes in a number of applications. For instance, fecal lactoferrin has been used as a marker for leukocytes to distinguish noninflammatory diarrhea from inflammatory diarrhea, as disclosed in U.S. Pat. No. 5,124,252 (the “'252 patent”). Noninflammatory diarrhea caused by agents such as rotavirus, Norwalk-like agents and cholera, typically causes minimal to no intestinal damage and patients respond readily to oral rehydration. Inflammatory diarrheas include those caused by enteric pathogens such as Clostridium difficile, Shigella species, Salmonella species, Campylobacter jejuni and Entamoeba histolytica and those that have no clearly defined infectious agent such as CD and UC. U.S. Pat. No. 5,124,252 discloses an in vitro test for fecal leukocytes which aids in distinguishing inflammatory from noninflammatory diarrhea. The '252 patent discloses testing fecal samples suspected of containing leukocytes with an assay that utilizes an antibody for lactoferrin to determine the presence of leukocytes in the fecal sample.
Human lactoferrin also has been used as a marker for diagnosis of inflammatory gastrointestinal disorders, colon polyp and colorectal cancer as disclosed in U.S. Pat. No. 5,552,292 (the “'292 patent”). However, neither the method of the '252 patent nor that of the '292 patent disclose utility in distinguishing IBS and IBD. The samples tested by the assay of the '252 patent are samples suspected of containing leukocytes. This suspicion is owed to the patient presenting with diarrhea. However, 25-50% of persons having IBD do not present with diarrhea and, thus, the '252 patent does not relate to diagnosing etiology in such patients. As for the '292 patent, the disclosed method utilizes a 1:100 sample dilution which does not allow for accurate quantitation of lactoferrin levels. Further, the '292 patent discloses using partial forms of molecules for testing and not total endogenous lactoferrin, again affecting the accuracy of the quantitation. The method of the '292 patent also does not relate to utilizing lactoferrmn levels to distinguish between IBD and IBS. The population tested in the '292 patent, while including persons with UC and CD, did not include persons having IBS. Therefore, there remains a need in the diagnostic industry for a noninvasive method for differentially diagnosing IBD and IBS which utilizes human lactoferrin as a marker.
Given that lactoferrin has been shown to be a good marker for fecal leukocytes, tests have been developed to aid physicians in determining the presence of fecal lactoferrin. One such test is the LEUKO-TEST®, manufactured by TechLab, Inc. of Blacksburg, Va. The LEUKO-TEST® is a latex agglutination test for detecting fecal lactoferrin. It is noninvasive and demonstrates active intestinal inflammation thus providing physicians evaluating patients with diarrhea with important information concerning the severity of any underlying bacterial infection.
Even though the LEUKO-TEST® is useful for evaluating gastrointestinal illnesses, the latex agglutination format provides some limitations. In large hospitals with a high volume of specimens, batching is preferred. A format such as ELISA is more useful for batching than latex agglutination and has the option of automation. It also may indicate severity of the disease and the effectiveness of medical treatments, by measuring the levels of fecal lactoferrin. In the case of IBD, a rise in fecal lactoferrin may provide an early indicator for active disease and the effects of medical treatments.
Currently, there are no known in vitro diagnostic aids to assist treating physicians, or other clinical personnel, in distinguishing between IBD and IBS. Accordingly, there remains a need for an in vitro diagnostic aid to assist treating physicians and other clinical personnel in distinguishing between these two commonly presenting diseases.