U.S. patent application number 14/245841 was filed with the patent office on 2014-10-30 for compositions and methods for assessing gastrointestinal health.
This patent application is currently assigned to GENOVA DIAGNOSTICS, INC.. The applicant listed for this patent is Patrick Hanaway, Jeffrey Ledford. Invention is credited to Patrick Hanaway, Jeffrey Ledford.
Application Number | 20140322728 14/245841 |
Document ID | / |
Family ID | 49181443 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140322728 |
Kind Code |
A1 |
Hanaway; Patrick ; et
al. |
October 30, 2014 |
Compositions and Methods for Assessing Gastrointestinal Health
Abstract
The present invention relates to kits designed for the
collection of stool samples and methods of analyzing those samples
for biological markers of maldigestion, inflammation, and
imbalanced gut flora.
Inventors: |
Hanaway; Patrick;
(Weaverville, NC) ; Ledford; Jeffrey; (Fletcher,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hanaway; Patrick
Ledford; Jeffrey |
Weaverville
Fletcher |
NC
NC |
US
US |
|
|
Assignee: |
GENOVA DIAGNOSTICS, INC.
Asheville
NC
|
Family ID: |
49181443 |
Appl. No.: |
14/245841 |
Filed: |
April 4, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13614888 |
Sep 13, 2012 |
|
|
|
14245841 |
|
|
|
|
12901967 |
Oct 11, 2010 |
8541180 |
|
|
13614888 |
|
|
|
|
Current U.S.
Class: |
435/7.9 |
Current CPC
Class: |
G01N 2333/44 20130101;
G01N 2333/96433 20130101; C12Q 1/04 20130101; G01N 33/56905
20130101; G01N 33/6893 20130101; G01N 33/68 20130101; G01N 33/573
20130101; G01N 2333/4727 20130101; G01N 2800/065 20130101 |
Class at
Publication: |
435/7.9 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A method of assessing a patient's gastrointestinal health, the
method comprising: (a) providing a stool sample from the patient;
and (b) subjecting the sample to three tests: a first test for
maldigestion, a second test for inflammation, and a third test for
gut flora.
2-23. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to kits designed for the
collection of stool samples and methods of analyzing those samples
for biological markers of maldigestion, inflammation, and
imbalanced gut flora.
BACKGROUND
[0002] Irritable bowel syndrome (IBS) is a functional disorder of
the intestine characterized by altered bowel function (diarrhea,
constipation, or both) and abdominal pain. It is the most common
gastrointestinal disorder seen in general practice, and the
prevalence of IBS has been estimated to be as high as 20% of the
general population of North America. The majority of IBS sufferers
are women, and women account for 80% of the cases of severe
IBS.
SUMMARY
[0003] The methods described herein offer a concise look at the
overall health of a patient's gastrointestinal (GI) tract. They
include a number of non-invasive tests that evaluate digestion,
inflammation, and gut flora. The outcome of these tests can assist
in the diagnosis and treatment of IBS. Accordingly, in one aspect,
the invention features methods of assessing a patient's
gastrointestinal health by providing a stool sample from the
patient and subjecting the sample to three tests: a first test for
maldigestion, a second test for inflammation, and a third test for
gut flora. Testing for maldigestion can include assessments of
digestion and/or absorption of nutrients from the GI tract. For
example, the first test for maldigestion can include testing for
pancreatic insufficiency by evaluating the amount (e.g., the
concentration) of a pancreatic elastase (PE), or a heterologous
protein that is co-regulated with PE, in the stool sample. The PE
can be the isozyme pancreatic elastase 1 (PE1). Concentrations of
PE1 that are more than about 200 .mu.g per gram of stool, but less
than about 350 .mu.g per gram of stool, indicate mild pancreatic
insufficiency; concentrations of more than about 100 .mu.g of PE1
per gram of stool, but less than about 200 .mu.g of PE 1 per gram
of stool, indicate moderate pancreatic insufficiency; and
concentrations of less than about 100 .mu.g of PE1 per gram of
stool indicate severe pancreatic insufficiency. When desired (e.g.,
when pancreatic insufficiency is moderate to severe), the methods
can also include a step of testing for intestinal permeability,
generating a comprehensive parasitology profile, and/or testing for
Celiac disease. When desired (e.g., when pancreatic insufficiency
is moderate to severe), the methods can also include a step of
assessing bone resorption, analyzing glucose and/or insulin levels,
testing for Celiac disease, and/or testing for bacterial overgrowth
in the small intestine.
[0004] The second test, for inflammation, can be carried out by
testing for calprotectin or occult blood in the stool sample. The
testing for calprotectin can be carried out by evaluating the
amount of calprotectin (e.g. its concentration), or a heterologous
protein that is co-regulated with calprotectin, in the stool
sample. Calprotectin elevated to about 50-120 .mu.g per gram of
stool indicates low-grade inflammation of the gastrointestinal
tract; calprotectin elevated to more than about 120 .mu.g per gram
of stool indicates significant inflammation in the GI tract; and
calprotectin elevated to more than about 250 .mu.g per gram of
stool indicates severe inflammation in the GI tract. Elevation to
over about 250 .mu.g/g also indicates that a patient with
inflammatory bowel disease is at high risk of relapse within one
year. Signs of inflammation can be analyzed further by assessing
intestinal permeability, testing for food allergies, and/or
performing a comprehensive parasitology profile.
[0005] The third test, to examine gut flora, includes testing for
harmful and imbalanced gut flora and preferably includes testing
for both harmful and non-harmful (e.g., non-pathogenic or
beneficial) gut flora. Harmful gut flora includes, but is not
limited to, Clostridium difficile and Helicobactor pylori;
imbalanced gut flora includes, but is not limited to, Klebsiella
species, Pseudomonas species, and Clostridial species; and
beneficial gut flora includes, but is not limited to, lactobacillus
species, bifidobacteria species, and Escherichia coli. Where there
is a presence of harmful bacteria, an excess of imbalanced gut
flora and/or insufficient beneficial gut flora, there is a
maldistribution of flora within the total community. The presence
of harmful bacteria, an excess of imbalanced flora and/or an
insufficiency of beneficial gut flora are all forms of dysbiosis.
Treatment for dysbiosis can include probiotic treatment, and when
the gut contains harmful gut flora or imbalanced flora, a patient
can also be treated with anti-microbial herbs and/or
antibiotics.
[0006] Regardless of outcome, the methods of the invention or a
step within the methods (e.g., testing for inflammation) can be
repeated at a later point in time (e.g., six weeks later). Thus,
the methods of the invention can be repeated periodically and used
to monitor a patient. The monitoring can determine whether a
treatment (e.g., a drug treatment) or lifestyle change (e.g., a
change in diet or exercise) is having a measurable effect on the
tested parameter(s).
[0007] In another aspect, the invention features kits that can be
used to provide stool specimens in a condition suitable for testing
in the manner described above. For example, a kit for the
collection of a stool sample can include (a) a collection tub; (b)
a tube containing a fixative medium; (c) a tube containing a medium
that maintains the relative proportions of organisms in a stool
sample; and (d) written materials. The fixative medium maintains
the integrity of organisms within the stool for analysis, and a
suitable example is SAF medium. The medium that maintains the
organisms (i.e., a medium that does not selective kill or
selectively support any given organism) can be Cary-Blair
medium.
[0008] The written materials can be presented in various forms and
can include one or more of: (a) instructions for use; (b) a
requisition form; and (c) a mailing envelope or other materials for
transporting the sample.
[0009] In addition to the components listed above, the kit can
include other items such as one or more of: (a) a holder to suspend
the collection tub over a toilet; (b) an empty cup; (c) an
absorbent pad; (d) a flat tool suitable for insertion into a
hand-held tube (e.g., a wooden stick such as a tongue depressor or
a similarly shaped item made from wood, plastic, or other
materials); and (e) a glove (e.g., a disposable glove, which may be
biodegradable).
[0010] An IBS diagnosis is based on identifying positive symptoms
consistent with the condition and excluding other conditions with
similar clinical presentations. IBS symptoms often mimic those
associated with other GI conditions, such as maldigestion and
disorders of absorption (e.g., celiac disease, lactose intolerance,
pancreatic insufficiency), infection and dysbiosis, as well as
inflammatory bowel disease. IBS is differentiated from IBD
(irritable bowel disease) in that, unlike IBD, IBS does not cause
severe inflammation, ulcers or other damage to the bowel. Where the
only diagnosis is a diagnosis of exclusion, the average time from
the onset of symptoms to a positive diagnosis of IBS is nearly
three years. In addition, incorrect symptom attribution may lead to
referral to a gastroenterologist and unnecessary procedures (such
as colonoscopy or endoscopy), hospitalization, or surgery (e.g.,
appendectomy, cholecystectomy, or hysterectomy). Currently, Rome
III criteria are used to diagnose functional gastrointestinal
disorders such as IBS, although these criteria are not
significantly utilized in primary care (as they do not
differentiate therapeutic choices). Diagnosis based on tests to
evaluate digestion, inflammation, and infection/gut microflora will
result in more timely and accurate diagnosis of IBS.
[0011] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic representation of a culture plate,
divided into quadrants, that has been inoculated and streaked using
the spreading pattern shown.
[0013] FIG. 2 is a flow chart depicting representative analytes and
pathogens that can be assessed to evaluate digestion, inflammation,
and gut flora in a patient.
[0014] FIG. 3 is a representative report illustrating the manner in
which information obtained in the methods of the invention can be
conveyed to a clinician. The report can be supplied physically or
by computer graphics.
DETAILED DESCRIPTION
[0015] The compositions and methods described herein can be used to
assess gastrointestinal health in virtually any patient, regardless
of age and regardless of whether or not the patient has any
specific symptom or complaint (i.e., the method can be carried out
on a patient in good health). In many cases, however, the patient
will be a person who is complaining of abdominal pain, perhaps
associated with gas or bloating, constipation or diarrhea, or other
symptoms of a gastrointestinal disease or disorder.
[0016] Chronic maldigestion can lead to bacterial or fungal
overgrowth and alterations in gut permeability. Toxins and large
molecules that escape the intestinal barrier can enter the general
circulation, inflame the liver, burden the body's detoxification
system, and increase the risk for food allergies, joint disease,
and imbalances in overall health. Malabsorption can lead to
deficiencies of nutrients, proteins, carbohydrates and fats. This
can result in long term health complications such as anemia,
malnutrition, impaired metabolism and other diseases, such as
osteoporosis. Chronic dysbiosis (a microbial imbalance) can lower
the levels of beneficial short chain fatty acids and alter
bacterial metabolic activity, thereby increasing the risk of
carcinogenesis, hormonal imbalance, and GI inflammation. Altered GI
immune function and exposure to bacterial pathogens can lead to
diarrhea, mucosal inflammation, intestinal permeability, toxin
production and autoimmune disorders. Although the invention is not
so limited, patients suspected of suffering from one or more of
these conditions (e.g., patients complaining of any of these
symptoms or exhibiting signs of any of these conditions) are
candidates for testing as described herein. Further, any of the
methods described herein can include a step of identifying a
patient as a candidate for testing (e.g., a patient complaining of
gastrointestinal distress or exhibiting signs of an associated
condition). For example, the methods can include a step of
questioning a patient about their gastrointestinal health and/or
performing a physical examination.
[0017] Digestion can be assessed by testing for pancreatic
sufficiency or insufficiency, as the case may be, which can, in
turn, be assessed by determining the level of expression or
activity of a protein that is produced primarily or exclusively by
the pancreas and that remains stable or detectable following
passage through the intestine. For example, one can assess a
pancreatic elastase (PE), for example PE1, or another marker of
pancreatic function (e.g., a protein that is co-regulated with
pancreatic elastase). Pancreatic elastases are produced by the
human pancreas and, as noted, are an indicator of exocrine
pancreatic function, as levels of PE are largely unaffected by
transit through the GI tract or enzyme supplementation. These
levels correlate with duodenal outputs of amylase, lipase, trypsin,
and chymotrypsin, which may also be assessed. PE levels from
100-200 .mu.g/g of stool are associated with moderate pancreatic
insufficiency, whereas values below 100 .mu.g/g indicate severe
pancreatic insufficiency. Clinically, decreased levels of PE in
stool reflect the need for exogenous digestive enzyme
supplementation to support exocrine pancreatic deficiency.
[0018] Expression and activity of PE can be assessed in numerous
ways using techniques known in the art. For example, PE expression
can be detected with proteins that specifically bind PE (e.g., an
anti-PE antibody). In a particular embodiment, PE expression can be
assessed using a commercially available test, such as the
ScheBo.RTM. PE1 kit (ScheBo Biotech, Giessen Germany). A random
stool sample can be collected according to the patient instructions
provided with the kit. Briefly, a 100 ml polypropylene container is
filled 1/4-1/3 full with stool by the patient and homogenized using
the tool (e.g., a wooden tongue depressor) provided. The container
is closed tightly, placed in a shipping device, and sent to the
laboratory. Stool samples should be received by the lab within 8
days of collection and are stable for three days thereafter when
stored at 2-8.degree. C. Frozen aliquots are set up in 1.5 ml
microcentrifuge tubes for longer storage at -20.degree. C. Of
course, samples may be provided otherwise. For example, samples may
be collected from hospitalized patients and taken directly to the
hospital's lab or a nearby testing facility.
[0019] In the ScheBo.RTM. PE assay, an ELISA plate is coated with a
monoclonal antibody that specifically binds human PE1. PE1 present
in a sample or standard is bound to the antibody and thereby
immobilized on the plate. A complex of monoclonal anti-PE1-biotin
and peroxidase (POD)-streptavidin binds to PE during the next
incubation. The peroxidase oxidizes ABTS
(2,2'-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid)diammonium
salt), which turns dark green. Finally, the concentration of
oxidized ABTS is determined photometrically. Polyclonal antibodies
can also be used to detect proteins such as PE1 that are produced
by the pancreas and stably transported through the gastrointestinal
tract.
[0020] As noted, PE1 serves to indicate pancreatic function (e.g.,
sufficiency or insufficiency). Where levels are greater than about
200 .mu.g/g of stool, exocrine pancreatic function is adequate and
no further action is necessary with respect to pancreatic function.
However, a physician may wish to consider pancreatic
supplementation if levels are in the low normal range (e.g.,
200-350 .mu.g/g). Healthy individual produce more than 500 .mu.g/g
of PE. Thus, levels between about 200 and 500 .mu.g/g suggest a
deviation from optimal pancreatic function. Where this outcome is
found, the clinician should consider digestive enzyme
supplementation if one or more of the following conditions is
present: loose, watery stools; undigested food in the stool;
post-prandial abdominal pain; nausea or colicky abdominal pain;
gastroesophageal reflux symptoms; bloating or food intolerance. Low
levels of PE1 (for example, .about.100-200 .mu.g PE/g of stool)
indicate mild to moderate pancreatic insufficiency and pancreatic
enzyme supplementation as well as further testing to assess
intestinal permeability and to determine the profile of intestinal
parasites is advised. Celiac testing can also be performed. Very
low levels of PE1 (for example, <.about.100 .mu.g PE per gram of
stool) indicate moderate to severe pancreatic insufficiency. In
this event, pancreatic enzyme supplementation and vitamin and
mineral supplementation are indicated.
[0021] In addition to treatments targeted to the pancreas,
physicians may wish to consider other medical tests and treatments.
For example, reduced PE is found in over 50% of type 1 diabetics
and 35% of type 2 diabetics. Diabetes secondary to exocrine disease
could be much more frequent than previously thought; studies have
shown that low PE is closely related to glycemic control. Exocrine
pancreatic function is also frequently impaired in gallstone
sufferers and post-cholecystectomy patients. There is a high
prevalence of phathological changes in exocrine pancreatic function
in patients with gallstones. Nearly one third of patients with
osteoporosis have reduced concentrations of PE. Vitamin D levels
may also be significantly decreased in these patients. PE is also
useful in monitoring exocrine pancreatic function caused by:
chronic pancreatitis, auto-immunopathies and connective tissue
diseases, chronic inflammatory bowel disease, and intestinal
malabsorption with mucosal atrophy. A transient reduction of PE can
occur with villous atrophy. After mucosal regeneration, PE levels
return to normal. PE can therefore be used as a monitoring tool in
patients with Celiac disease and other malabsorptive conditions.
Accordingly, the present methods in which digestion is assessed by
examining PE can be carried out periodically to monitor a patient's
condition and/or recovery, and the methods can be expanded to
include other tests such as those mentioned above. For example,
further testing can include one or more of: bone resorption
assessment, glucose/insulin analysis, Celiac testing, and testing
for bacterial overgrowth in the small intestine.
[0022] Another way to assess digestion is by evaluating stool alpha
1 chymotrypsin levels or assessing fecal fat content. Chymotrypsin
is an enzyme secreted by the pancreas that functions in protein
digestion. Levels of stool chymotrypsin can be determined using a
stool sample that is collected as described herein and preserved in
a minimal microbial growth media. A high salt surfactant can be
used to release the chymotrypsin contained within the sample. The
reaction of the free chymotrypsin with a spectrophotometrically
active substrate facilitates the determination of chymotrypsin
activity in the sample. Results can be expressed as units of
chymotryptic activity relative to the grams of stool analyzed. A
normal result for this assay is .about.0.9-26.8 U/g stool. Low
levels of chymotrypsin (<0.9 U/g) indicate exocrine pancreatic
insufficiency. Therapy should include exogenous supplementation of
pancreatic enzymes including lipase. Elevated levels of
chymotrypsin (>26.8 U/g) suggest a rapid transit time
(diarrhea). A faster transit time reduces the intestinal
degradation of chymotrypsin, which results in an increased recovery
of this enzyme. Chymotrypsin could also be elevated with excess
pancreatic enzyme supplementation.
[0023] As both pancreatic elastase and chymotrypsin are indicators
of exocrine pancreatic function, the clinical indications for PE
also apply to cymotrypsin. Therefore, the methods in which
chymotrypsin is assessed can also include a step of identifying a
patient who has: loose, watery stools, undigested food in the
stool, post-prandial abdominal pain, nausea or colicky abdominal
pain, gastroesophageal reflux symptoms, or bloating or food
intolerance. Other conditions associated with reduced chymotrypsin
include diabetes, cystic fibrosis, chronic pancreatitis, and
malabsorption.
[0024] Both chymotrypsin and PE are highly accurate in
distinguishing between pancreatic maldigestion and intestinal
malabsorption (82% and 92%, respectively). PE is not affected by
bovine or porcine enzyme supplements, so patients do not have to
discontinue therapy to assess baseline levels. Chymotrypsin is
affected by exogenous supplementation, which makes it an ideal tool
to monitor dosing adequacy. When chymotrypsin values fall within
the reference range in supplemented individuals, the clinician can
be confident that an appropriate dosage of digestive enzymes is
being administered. As noted, the present methods can be carried
out not only in the context of an initial presentation and
diagnosis, but also in the context of ongoing monitoring. While PE
may be a more accurate non-invasive marker to assess exocrine
pancreatic function, chymotrypsin is the preferred marker to
monitor enzyme supplementation.
[0025] Elevated levels of fecal fat (e.g., elevated cumulative
levels) are also an indicator of maldigestion. Fecal fats include
triglycerides, long chain fatty acids (LCFAs), cholesterol and
phospholipids, which can be extracted with methods known in the art
and measured individually to obtain a measure of fecal fat levels.
To evaluate fecal fat, a stool sample can be submitted in a
preservative (e.g., 5% formalin), and lipids can be extracted from
the sample using an organic extraction. Once the stool lipids are
isolated, standard automated chemistry assays can be used to
determine the levels of the individual fecal fat components,
including triglycerides, cholesterol, phospholipids and long chain
fatty acids. Cumulative totals of these components outside the
range of 2.6 to 332.4 mg/g stool are indicative of malabsorption
Triglycerides represent the major component of dietary fat (on
average 120 g of a 125 g daily load). Elevated levels are
suggestive of incomplete fat hydrolysis, which can be caused by
exocrine pancreatic insufficiency or bile acid insufficiency.
Elevated triglycerides with normal LCFAs have been noted in
patients with steatorrhea due to pancreatic insufficiency.
Triglycerides may also be elevated with rapid transit time, which
impairs the breakdown and absorption of these lipids.
[0026] LCFAs are normally readily absorbed in a healthy mucosa.
Elevated levels suggest malabsorption, reduced pancreatic function
or bile insufficiency. Increased LCFAs have also been noted after
acute intestinal infections.
[0027] Fecal cholesterol is derived from the diet, bile, and from
mucosal epithelial break-down. In a healthy gastrointestinal tract,
about 40-60% of dietary cholesterol will be absorbed. Elevated
levels are a reflection of mucosal malabsorption. Impaired
absorption of fecal cholesterol occurs in Celiac disease secondary
to damaged mucosa from gluten ingestion.
[0028] Phospholipids are derived from three specific sources: bile
(50%), diet (25%), and mucosal desquamation (25%). The major
dietary-derived phospholipids include phosphatidyl choline,
phosphatidyl serine, phosphatidyl ethanolamine and cardiolipin. In
a healthy individual, nearly 85% of intestinal phsopholipids are
absorbed. As phospholipids are derived from more than one source,
elevations could occur from the following: malabsorption,
inadequate bile salt resorption, or increased mucosal cell
turnover. Referring to other absorptive markers can help determine
the cause of high fecal phospholipids.
[0029] The following conditions can contribute to the impaired
absorption of fecal fat: pancreatic insufficiency (specifically
lipase), cholestasis (e.g., bilary obstruction or liver disease),
interrupted enterohepatic circulation (e.g., ileal disease or bile
sale deconjugation from small bowel bacterial overgrowth), Celiac
disease, short bowel syndrome, and Whipple's disease (rare). In
general, an elevation in any one of the fecal fat markers is
suggestive of malabsorption (see above for the clinical
significance of individual absorptive markers).
[0030] Inflammation affecting the GI tract can be detected in a
variety of ways, including by tests for calprotectin expression and
occult blood. Calprotectin is a calcium-binding protein found
primarily in neutrophils that serves as a direct marker of
intestinal inflammation. Infection, tissue damage, or increased
permeability of the mucosa results in the migration of granulocytes
into the intestinal lumen. As with PE, calprotectin can be detected
by methods known in the art, including methods in which a
calprotectin-specific binder is used to detect expression. For
example, one can use the PhiCal.RTM. quantitative EIA (Calpro
AS).
[0031] Calprotectin is a useful analyte because it is resistant to
bacterial degradation in the gut and is stable in stool for up to
one week at room temperature. It is also unaffected by medications,
dietary supplements, and enzymatic degradation. Further,
calprotectin: reflects the flux of leukocytes into the intestinal
lumen; is released upon activation and degranulation of
neutrophils; correlates strongly with 111-indium-labeled leukocyte
excretion as well as histologic and endoscopic grading of disease
activity in ulcerative colitis; helps differentiate between IBS and
active IBD; predicts relapse in patients with IBD and serves as an
objective marker to assist in treatment protocols; and assists in
selecting patients for endoscopy and in monitoring responses to
treatment (especially in children who may require general
anesthesia to undergo more invasive analyses).
[0032] For calprotectin analysis, a random stool sample is
collected according to patient instructions for the calprotectin
PhiCal.RTM. test. Briefly, a suitable container (e.g., a 100 ml
polypropylene tube) is filled 1/4-1/3 full with stool by the
patient and homogenized using a tool, such as a wooden tongue
depressor. Controls for low and high calprotectin expression can be
assayed with each batch of samples. PhiCal.RTM. uses a polyclonal
antibody against calprotectin that is absorbed to the surface of a
plastic well to bind calprotectin in diluted stool samples. As the
antibody is linked to an enzyme, a subsequent reaction in which the
enzyme catalyzes a reaction to yield a detectable (e.g.,
fluorescent or colored) product provides an easy means for
detecting and quantitating bound calprotectin. Values below
.about.50 .mu.g of calprotectin per gram of stool are not
indicative of inflammation in the GI tract, and no further action
is necessary based on this result. Values between 50-120 .mu.g/g
stool are associated with low-grade inflammation, which could be
due to post-infectious irritable bowel syndrome (IBS), infection,
food allergies, polyps, neoplasia, non-steroidal anti-inflammatory
drugs (NSAIDs) or IBD in remission. It is prudent in these cases to
repeat the calprotectin assay after about six weeks. If levels
remain elevated after ruling out other etiologies, further
investigative tests (e.g., endoscopy or imaging) should be
considered. These further tests include: stool culture, an
intestinal permeability assessment, a food antibody assessment, and
a comprehensive parasitology profile. Therapeutic intevention
includes probiotics, fish oils, N-acetylglucosamine, and/or rutin.
Calprotectin expression above .about.120 .mu.g/g indicates
significant inflammation, possibly caused by IBD, infection, food
allergies, NSAID use, polyps, adenomas, colorectal cancer, or
diverticulitis. Unless the source of the inflammation is clear,
further evaluation is recommended and may include endoscopy and/or
colonoscopy. One can also assess microbiology/parasitology at this
stage, and further testing includes any of the tests listed above
in connection with lower elevation of calprotectin. Where
calprotectin is severely elevated (>250 .mu.g/g), there is a
strong likelihood that the patient has active IBD or are at a high
risk of relapse to active IBD within one year. In this event, IBD
should be managed with standard therapies. Intervention can include
administration of probiotics, fish oils, N-acetylglucosamine,
rutin, and/or anti-inflammatory agents (e.g., leukotriene
inhibitors and TNF-alpha antagonists). For patients with IBD,
calprotectin levels between 250-500 .mu.g/g indicate low to
moderate disease activity. Levels above 500 .mu.g/g suggest high
disease activity. Patients with IBD in remission and levels above
250 .mu.g/g have a high risk of relapse within one year.
[0033] Where calprotectin levels are above 120 .mu.g/g, there is
likely significant inflammation in the gastrointestinal tract. The
cause could be IBD, infection, NSAID use, polyps, adenomas, or
colorectal cancer. Calprotectin may also be elevated in children
with chronic diarrhea secondary to cow's milk allergy or multiple
food allergies. Further procedures can be carried out to determine
the cause more specifically. Levels ranging from 50-120 .mu.g
indicate less severe inflammation. Values below 50 .mu.g/g indicate
there is little or no inflammation in the gastrointestinal
tract.
[0034] Regardless of the cause, patients with elevated calprotectin
should be tested further with, for example, an intestinal
permeability assessment, an allergy antibody assessment, Celiac
panel, ImmunoGenomic.TM. profile, or a comprehensive parasitology
profile.
[0035] Another marker for inflammation is an eosinophil protein
(e.g., eosinophil protein X or EPX, also known as
eosinophil-derived neurotoxin (EDN)). In healthy individuals,
eosinophils reside in the connective tissue layer of the gut, known
as the lamina propria. It is not until damage occurs to the lamina
propria that eosinophils migrate into the gut lumen. Eosinophils
contain a number of highly cationic proteins such as eosinophil
cationic protein, major basic protein, eosinophil peroxidase, and
EPX. Upon eosinophil degranulation, these cationic proteins are
release and can be assayed. As the proteins possess cytotoxic
properties, their accumulation is associated with inflammation and
tissue damage.
[0036] While assessing eosinophilic activity is informative, tests
that require colonoscopy or biopsy have limited utility for
office-based practitioners. Eosinophil markers in stool, however,
can be assayed according to the present methods as indicators of
inflammation. As with other markers described herein, eosinophilic
markers can provide information regarding a patient's initial
treatment and can be assayed over time to monitor that treatment.
For example, baseline levels of EPX can be used to determine
intestinal inflammation associated with food allergy and to monitor
dietary changes. Studies have demonstrated a significant reduction
in EPX after three months on a successful elimination diet.
[0037] Assays for EPX can be based on extraction of EPX from neat
stool using a cationic extraction buffer. Once extracted, the
protein can be detected and quantitated using any EPX-specific
binding protein (e.g., an antibody). Eosinophils are bone-marrow
derived cells that secrete a range of highly toxic granule proteins
and other inflammatory mediators. The cells are characterized by
the presence of dense granular deposits in their cytoplasm that
contain proteins that mediate the inflammatory response and tissue
damage. EPX is one of the highly cationic proteins contained within
these granules. Eosinophils play a role in allergy and in the
response to parasites and are found in significant numbers in the
underlying connective tissue of the respiratory, gastrointestinal,
and urogenital tracts. Biopsies of patients with IBD (ulcerative
colitis and Crohn's disease) or Celiac disease have demonstrated
marked infiltration of eosinophils. This infiltration plays a role
in the pathogenesis of the inflammatory processes associated with
these disease states. Further, increased levels of EPX have been
demonstrated in the feces of patients with active disease.
[0038] If desired, EPX can be evaluated using the EDN kit available
from the Medical and Biological Laboratories Co., Ltd (MBL). Once
extracted, EDX is quantified using a sandwich ELISA. The reportable
range of the assay is 0.9 ng/ml to 40.0 ng/ml. Where EPX is present
at <.about.7.0 .mu.g/g, there is no active inflammation of the
GI tract. However, elevated levels of EPX (>.about.7.0 .mu.g/g)
indicate inflammation and/or tissue damage in the GI tract.
Underlying causes include food allergy and/or atopic dermatitis,
protein-sensitive enteropathy, helminthic infection, IBD, allergic
colitis, chronic diarrhea, chronic alcoholism, bowel cancer,
eosinophilic gasteroenteritis (rare), and gastroesophageal reflux.
Increased levels of EPX have also been found in ulcerative colitis
and Crohn's Disease, with elevations correlating with disease
activity. As a non-invasive marker, EPX offers increased
sensitivity for evaluating inflammatory disease activity and for
predicting relapses in patients with IBD.
[0039] Occult blood is blood in the stool that is present in
amounts too small to be seen but large enough to be detected by
chemical tests. In our methods, we test for occult blood as an
indicator of inflammation and a sign of certain gastrointestinal
disorders. Occult blood can be detected in several ways, including
by immunoassays for hemoglobin. For convenience, one can use a
commercially available test such as the Hemosure.RTM. One Step
Immunological Fecal Occult Blood (iFOB) test supplied by Quidel
Corporation. This test is a rapid, qualitative, sandwich dye
conjugate immunoassay for the detection of human hemoglobin in
feces. It employs a unique combination of monoclonal and polyclonal
antibodies to selectively identify hemoglobin in test samples with
a high degree of sensitivity. In less than five minutes, elevated
levels of human hemoglobin as low as 0.05 .mu.g hHb/ml can be
detected and positive results for high levels of hemoglobin can be
seen in the test as early as two to three minutes. Stool samples in
Cary-Blair medium can be tested in this assay within about seven
days, and stools collected in the preservation buffer tube are
stable as follows: for six days at 37.degree. C., six months at
4.degree. C., and 20 months at -20.degree. C. A monoclonal
antibody-based assay that can be used is the Hemosure.RTM. test,
which is specific for human hemoglobin.
[0040] A positive result can indicate ulcers (e.g., peptic ulcers),
polyps, diverticulitis, IBD, or colorectal cancer. Bowel lesions,
polyps and colorectal cancers may not bleed or may bleed
intermittently, however, and a test result can be negative even
when disease is present for this reason. Repeated testing is
advisable if symptoms persist, and physicians are likely to order
repeated testing for occult blood as well as other, invasive tests
(e.g., colonoscopy) or imaging (e.g., a barium enema) whenever
colorectal cancer is suspected. Patients should be cautioned not to
take vitamin C supplements for several days prior to testing for
occult blood, as ingestion over about 250 mg of vitamin C per day
inactivates the test.
[0041] Conversely, a test can be positive in healthy patients
because certain medications may cause gastrointestinal irritation
resulting in occult bleeding. These include rectal suppositories
and oral medications such as aspirin and corticosteroids. If a
patient has not refrained from eating undercooked meats, a false
positive result may ensue. However, the Hemosure.RTM. iFOB test is
highly specific and should not react to hemoglobin from fish, beef,
chicken, rabbit, or goat. Stool samples may also become
contaminated with blood if collected during menstrual bleeding or
if a patient has blood in the urine. Positive and negative controls
should be run with each test. The positive control can be diluted
human blood, and the negative control can be a known blood-free
sample, such as water or a buffered solution.
[0042] Accordingly, the present methods can include repeated occult
blood tests, positive and negative controls for occult blood, and a
step of obtaining information from a patient regarding vitamin C
intake, meat intake, and menstruation.
[0043] Where the present methods assess inflammation, they can also
include an assay for lactoferrin. Human lactoferrin is an 80
kilodalton glycoprotein that binds iron and is secreted by most
mucosal membranes. It is a major component of polymorphonuclear
neutrophils (PMNs), which are the primary component of an acute
inflammatory response.
[0044] Assessing inflammation is especially useful in
distinguishing IBS from IBD, as there is little if any inflammation
associated with the former condition.
[0045] Lactoferrin can be assessed in a number of ways, including
the IBD-Chek.TM. test, which uses antibodies to human lactoferrin.
The microtiter wells contain immobilized polyclonal antibody
against lactoferin. The detecting antibody consists of polyclonal
antibody conjugated to horseradish peroxidase. In the assay an
aliquot of fecal specimen is emulsified in the diluent and
transferred to a microtiter well. Any lactoferrin present in the
sample binds to the immobilized antibody. After incubation, the
wells are washed and the conjugated antibody is added. A second
wash step removes unbound conjugate. With the addition of
substrate, any conjugate present as the result of bound lactoferrin
results in color formation, which can be quatitated with a
spectrophotometer. At 450/620 nm, a negative result is <0.160, a
positive result is .gtoreq.0.160. All positive results should be
confirmed by repeat testing.
[0046] In the paragraphs that follow, we discuss methods for
assessing gut flora.
[0047] Identification of microorganisms is key to determining
microbial imbalance due to redistributions within the microbiome,
parasitic infections, and other changes in gut flora. Traditional
culture methods enable the identification and semi-quantitation of
specific organisms through the utilization of differential growth
media. This can also be accomplished using automated biochemical
instruments such as Vitek. Other methods and techniques that can be
used include mass spectrometry methods such as MALDI-TOF-MS
(Matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry). The methodology uses species- and strain-specific
biomarkers to identify organisms. Microarray technology can also be
used to evaluate nucleic acids such as 16sRNA, small peptides or
molecules such as toxins that are specific for the microorganism,
Carbohydrate (e.g., polysaccharide) profiles can also be
determined. Nucleic acid probe technology can also be used in
combination with fluorescent microscopy for microbial
quantification and identification. Other methods for assessing the
microbiome include nucleic acid amplification by PCR and RNA or DNA
sequencing.
[0048] General stool culture procedure: Methods suitable for use in
culturing or otherwise treating stool samples are known in the art
and can be used in the context of the present invention. In the
following paragraphs, we provide information regarding the
processing, testing, and interpretation of stool bacterial
cultures. These processes can be used to generally evaluate the
microbial community rather than to diagnose infectious disease by
analyzing pure cultures of specific organisms.
[0049] The microbial community that resides in the gastrointestinal
tract is comprised of numerous species of naturally occurring
bacteria. While some defined organisms are clearly harmful, most
bacteria that typically found in the GI tract are beneficial and
are present in numbers that promote good health. More specifically,
beneficial bacteria, such as lactobacilli and bifidobacteria, play
an important role in promoting a healthy gut microflora environment
and ensuring proper digestion. These organisms can prevent the
over-colonization of the gut with pathogenic organisms and may
reduce the risk of certain gastrointestinal diseases. The role of
some commensal organisms may vary depending on their presence as a
percentage of the total bacterial population. Using the present
methods, one can evaluate the bacteria present in a stool sample in
a more comprehensive way. Identifying each organism and enumerating
it relative to other isolates provides the clinician with a picture
of the microbial community present in the GI tract and, more
specifically, the colonic environment, at the time the sample was
collected. This information is useful in determining whether the
patient is suffering from clinical gastritis or subtle, atypical
aberrations that may indicate a sub-clinical disease process. The
bacteria present can be categorized as harmful (or pathogenic),
potentially harmful (or pathogenic), or beneficial.
[0050] Several factors may affect the composition of the colonic
flora, including diet, transit time, stool pH, age, microbial
interactions, colonic availability of nutrients, bile acids, and
sulfate, as well as the ability of the microbes to metabolize these
substrates.
[0051] The predominant beneficial flora in the large intestine are
the bifidobacteria, which constitute as much as 25% of the overall
colonic flora in healthy adults. Recovery of these organisms from
the colon should therefore ideally be in the 3+ or 4+ ranges. In
the colon, obligate anaerobes such as bifidobacteria predominate
over facultative anaerobes such as lactobacilli by 1000:1. It is
for this reason that lactobacilli growth as low as 1+ or 2+ is
considered normal in healthy adults. Non-pathogenic E. coli
populate the distal colon, although they are usually found in
reduced quantities, comparable to levels of lactobacilli. A 1+ to
2+ concentration of non-pathogenic E. coli is therefore considered
normal.
[0052] Organisms of the genera Salmonella, Campylobacter, and
Shigella have been associated with the production of a broad
spectrum of clinical symptoms observed in the event of bacterial
gastritis. Accordingly, these organisms are designated as pathogens
and their presence normally results in acute diarrhea. Other
traditionally recognized intestinal pathogens include
enterotoxigenic E. coli, Shigella, Yersinia, Pleisiomonas, Vibrios,
Aeromonas, Campylobacter, the viral pathogen rotavirus, and the
parasites Cryptosporidium and Coccidia. A number of other bacteria
have been associated with gastrointestinal discomfort, but their
etiologic role is still largely undetermined.
[0053] Organisms such as Citrobacter freundii, Enterobacter
cloacae, and Klebsiella pneumoniae are usually classified as
"normal" flora. However, some studies have associated these and
other organisms with various gastrointestinal complaints when they
are the predominant organisms identified on stool culture in the
absence of other frank pathogens. The presence of traditionally
non-pathogenic bacteria in predominating numbers could indicate a
dysbiotic state in the colon. These organisms may be the direct
cause of a gastrointestinal disturbance, they may be aggravating
such a disturbance, or they may simply be present as an indicator
of some other disruptive process. Accordingly, we tend to classify
these organisms as potential pathogens. Yeast have also been
associated with clinical syndromes related to dysbiotic colonic
environments. There are no definitively recognized pathogenic
yeast, and we therefore refer to yeast as a potential pathogen when
present in elevated numbers. Candida albicans is the most
significant isolate of yeast, and its presence is associated with a
broad spectrum of clinical conditions.
[0054] Beneficial bacteria are organisms whose presence in
substantial numbers has been associated with a healthy colonic
environment. These bacteria include Bifidobacterium sp.,
Lactobacillus sp. and Escherichia coli.
[0055] Useful materials include: stool transport (Cary-Blair
media); blood agar plates; Maconkey agar plates; colistin-nalidixic
acid (CNA) plates; hektoen-enteric agar (HE) plates; oxyrase
bidifobacter agar plates; candida ID agar plates; sterile swabs;
gloves; isoplater instrument (for automated streaking); and an
incubator.
[0056] Samples arriving for testing may be in a transport media
that preserves stool bacteria. Preferably, the transport media
suspends active metabolism but preserves viability. A suitable
medium is the Cary-Blair formula manufactured by the MML company.
Samples preserved in this medium can be reliably tested up to six
days after they have been collected from a patient.
[0057] To inoculate plates, the sample can be mixed before using a
sterile swab to transfer a sample of stool onto the surface of a
culture plate (e.g., to about a 0.5.times.0.5 cm patch). To assess
bacteria, one can inoculate one or more plates holding the
following culture media: blood agar (BAP), Hektoen-enteric agar,
Maconkey (MAC) agar, colistin-nalidix acid (CNA), candida ID agar,
and MCA bifidobacter agar. The sample can then be further
distributed on the surface of the agar either manually (e.g. using
a sterile loop or needle) or by an automated streaker instrument.
Typically, the MCA bifidobacter agar and the candida ID agar are
manually streaked. The plates are then incubated (e.g., at
30-37.degree. C.) for a number of hours (e.g., 8-24 hours or more)
prior to evaluation. Some plates (e.g., the bifidobacter agar
plates can be incubated longer (e.g., at 35.degree. C. for at least
or about 72 hours). Candida ID agar plates can be incubated for at
least or about 72 hours at 35.degree. C.
[0058] Following incubation, the plates can be assessed in numerous
ways. For example, one can begin by noting changes in morphology.
The CNA plate can be evaluated for alpha-hemolysis (green),
gamma-hemolysis (no hemolysis) and/or beta-hemolysis (clearing of
agar immediately surrounding a colony). Isolates can be recovered
from this plate and others. Lactobacillus, Streptococcus, and
Staphylococcus are a few of the isolates that may be recovered from
this plate. The HE agar plate can be examined for the presence of
lactose (yellow) and non-lactose fermenters, hydrogen sulfide
producers (black pigment) and clearly mucoid colonies. These plates
are useful in isolating Salmonella, which produce hydrogen sulfide,
and Shigella, which do not ferment lactose and appear as clear
colonies. Maconkey agar can be used to identify gram-negative
organisms. Almost all enteric bacilli will grow on this media.
Lactose fermenting colonies (pink), non-lactose fermenting colonies
(grayish or colorless), and mucoid colonies may appear on these
plates. The blood agar plate can be compared to the other media.
Swarming or beta-hemolysis that is present here but not on other
plates should be pursued.
[0059] In addition to gross observations regarding morphology, one
can identify and quantitate each organism. Growth quantitation is
important for determining the significance of the organism.
Referring to FIG. 1, organisms can be categorized according to the
extent of their growth. For example, organisms growing in the first
quadrant only can be designated "1+". Growth in the first and
second quadrants would be categorized as "2+", and so forth. These
determinations may be somewhat subjective. For example, if
organisms on the plate are clearly isolated, and there is one
discernable colony in quadrant 1 and 1 discernable colony in
quadrant 2, the quantitative report could be very few colonies or
1+ growth. However, if the reader is unable to determine the amount
present in quadrant 1 (due, for example, to heavy growth of
additional organisms), the outcome may be 2+ instead.
[0060] Affirmative identification of an organism can be carried out
in several ways. Procedures for many different biochemical and
media-based differential tests are known in the art. It is
generally true that, regardless which test is used, the most
accurate identifications are made when working with a pure culture.
Thus, we stress the advantages of working on a single,
well-isolated colony.
[0061] After quantifying and identifying the pathogens, one can
assess minimum inhibitory concentrations or perform botanical
assays. When indicated (e.g., when requested by a patient's
physician), sensitivities and botanical assays can be performed on
significant gram-negative enteric and non-enteric isolates (e.g.,
Staphylococcus aureus, species of bacillus, and yeast isolates.
[0062] In any of the present methods, the original sample can be
aliquoted, and portions can be subjected to ancillary testing.
[0063] Identifying bacteria: Any method known in the art can be
used to identify a given bacterium in stool samples. For example,
the present methods can include a step of identifying Clostridium
difficile, and this may be done through identification of a toxin
produced by these organisms. C. difficile is a gram-positive,
anaerobic, spore-forming bacillus that is the most commonly
identified cause of antibiotic-associated diarrheal disease. When
broad-spectrum antibiotic use suppresses the normal intestinal
flora, selective pressure allows toxigenic strains of C. difficile
to multiply. The toxins produced by toxigenic C. difficile strains,
toxin A and toxin B, have enterotoxic and cytotoxic effects,
respectively. Disease may vary from mild diarrhea to a condition
known as pseudomembranous colitis (PMC), which can be fatal if not
treated.
[0064] C. difficile organisms may not cause symptoms in 2-3% of
healthy adults, 20-30% of hospitalized patients, and 50% of
children under two years old. Thus, culture alone may be inadequate
to document C. difficile disease.
[0065] C. difficile antigens, including toxin A and toxin B, can be
detected with specific binding proteins (e.g., antibodies).
Commercially available assays, such as the ProSpecT.RTM.
Clostridium difficile Toxin A/B Microplate Assay, can be used for
convenience.
[0066] Other intestinal bacteria that can be assessed include
Helicobacter pylori, which may be of particular note due to its
association with gastritis, gastric ulcers, and stomach cancer.
Virulence factors include a vacuole-promoting cytotoxin and a
potent urease enzyme, which creates an alkaline microenvironment
that may allow continued growth despite stomach acidity. Typically,
infected hosts mount an inflammatory response that accounts for
much of the tissue damage. This organism can be harbored in a
"colonized" state in which no symptoms of gastric disease are
present.
[0067] H. pylori is a small, curved, gram-negative bacillus. It is
oxidase, urease, and catalase positive. It is slow-growing on
culture and requires specific conditions for growth. These features
can be exploited in testing for the presence of H. pylori in a
sample. Identification can also be made through urea-breath
testing, serological tests, and with gastric biopsy tissue.
Commercially available kits for detecting H. pylori include the
Premier Platinum HpSA enzyme immunoassay. This is a qualitative
assay for detecting H. pylori antigens in human stool. It employs a
plurality of monoclonal anti-H. pylori capture antibodies adsorbed
onto microwells. Diluted patient samples and a
peroxidase-conjugated antibody are added to the wells, and any
unbound conjugated antibody is subsequently washed off. Enzyme
substrate is added to produce a colored reaction product that can
be detected visually or spectrophotometrically.
[0068] Identification of protozoa: To assess altered gut flora, one
can also test stool samples for protozoa. For example, fecal slides
(e.g., stained fecal slides) can be used to identify intestinal
protozoan cysts, ova, and trophozoites. Slide-based procedures are
advantageous in that the slides are semi-permanent; they can be
preserved for re-inspection and can be shipped (e.g., to experts
for consultation). While protozoa can also be detected by direct
smear and concentration techniques, slide preparations are
generally considered to be more reliable for detecting protozoa.
For example, the slides can be prepared so they include areas of
both thinner and thicker stool density, which is optimal for
recovery and detection of parasites.
[0069] Stool samples can be prepared in several ways. While single
samples can be tested, it may be preferable to test multiple
samples (e.g., 2-4 samples) that were collected on different days
or to pool multiple samples from the same patient (e.g., three
samples collected over the course of about one week). The samples
can be stored in SAF preservation medium (sodium acetate, 10%
formalin), homogenized (e.g., by vortexing) and aliquotted. One or
more aliquots can then be filtered and concentrated by
centrifugation. The supernatant is discarded, and the resulting
sediment is analyzed. Samples placed in SAF medium should be stable
for about 60 days at room temperature.
[0070] By way of illustration, multiple stool samples from a single
patient can be concentrated by: (1) removing the caps from each
patient sample; (2) pouring an aliquot from each of the patient's
sample vials into a clean, appropriately labeled, empty vial and
mixing thoroughly; (3) capping the single vial including the mixed
samples; (4) suspending the sample; (5) centrifuging the sample;
(6) discarding the supernatant; and (7) reconstituting the pelleted
sample. This procedure can be carried out using SpinCon.RTM. tubes
(Meridian Diagnostics, Inc.) and Para-Pak Ultra.RTM. caps (Meridian
Diagnostics, Inc.). In that event, the procedure can be carried out
by: (1) removing the caps from each patient sample; (2) pouring an
aliquot from each of the patient's sample vials into a clean,
appropriately labeled, empty vial and mixing thoroughly; (3)
capping the single vial including the mixed samples with a Para-Pak
Ultra.RTM. filtration cap, removing the outer cap with the key
provided, and breaking off the tab located inside the cap; (4)
placing a SpinCon.RTM. tube onto the Para-Pak Ultra.RTM. cap,
inverting and tapping the tube gently on the counter to allow
approximately 3-5 ml of specimen to filter through the cap into the
SpinCon.RTM. tube; (5) removing the SpinCon.RTM. tube from the cap
and placing a plug cap on the tube; (6) centrifuging the tube
(e.g., at about 500.times.g for 10 minutes); (7) removing the tub
from the centrifuge and removing the top half of the tube; (8)
removing half of the supernatant with a disposable plastic pipette
from the bottom half of the SpinCon.RTM. tube and placing it in a
13.times.75 mm, appropriately labeled tube for EIA procedures; (9)
reconstituting the sample by vortexing and pouring roughly half of
the reconstituted sample into a clear polystyrene tube; and (10)
recapping the bottom half of the SpinCon.RTM. tube and capping with
a fresh plug cap. See also, Ash and Orihel, "Parasites: a guide to
laboratory procedures and identification", ASCP Press 45-7-013-00
ISBN: 0-89189-231-1, 1987; Yang and Scholten, Am. J. Clin. Path.
67:300-304, 1977; and Meridian Diagnostics, Inc., package inserts
for SpinCon and Para-Pak Ultra filtration devices, Rev. 2/01.
[0071] A suspension of a concentrated stool sample can be applied
to a standard microscope slide along with Lugol's (5%) iodine as a
contrast agent. Parasite ova and cysts are detected microscopically
and identified by morphology (e.g., shape, size, number of nuclei,
cell structure, and other morphological attributes). PVA-preserved
specimens are not recommended for iodine-stained preparations, but
concentrated SAF or formalin-preserved stool can be used.
[0072] By way of illustration, one can prepare a slide by: (1)
inserting a pipette into the tube or other vessel containing a
stool sample and mixing; (2) removing a sample with the pipette and
placing about one drop onto a plain microscope slide; (3) adding
about one drop of iodine solution to the drop of sample; (4) mixing
the iodine and stool sample with the corner of a glass coverslip;
and (5) placing a coverslip over the sample. The preparation should
be reasonably transparent. If it is too thick, it can be diluted
with saline before the coverslip is placed. By microscopic
examination, one can detect helminthes, ova, or larvae, and amoeba.
Artifacts such as white blood cells and red blood cells may also be
noted.
[0073] Giardia cysts are typically 8-19 .mu.m in diameter.
Enterobacter (e.g., E. coli) cysts have eight or more nuclei and
are 15-25 .mu.m in diameter. Chilomastix cysts are lemon-shaped,
uni-nucleate, and 6-10 .mu.m in diameter.
[0074] For trichrome slide preparation, samples can be concentrated
(e.g., by the process described above) and prepared for staining by
making a 1:1 dilution of the reconstituted sample and PVA in a tube
(e.g., a polystyrene tube). The sample is then poured from the tube
onto a paper towel, which absorbs the PVA but not the sample. The
sample can then be transferred to a microscope slide using an
applicator. Moving the applicator in a back-and-forth motion while
rolling allows for a varied distribution of material on the slide,
including thicker and thinner areas for examination. After transfer
to the slide, the sample can be left to dry at room
temperature.
[0075] Batches of samples prepared for trichrome staining and
examination can be run with positive and negative controls. For
example, two control slides can be stained with each batch of
patient specimens; a negative control slide can be made from a
sample known to be free of parasitic organisms and a positive
control slide can be made from a sample known to contain protozoan
cysts and trophozoites.
[0076] Trichrome staining is a rapid staining procedure that
provides excellent differentiation of internal structures of
intestinal parasites and separation of the organisms from
background material. It is also rapid, easy, and suitable for fecal
smears. The stain itself is the Wheatley modification of Gomori's
trichrome stain for tissue sections, and it is highly stable. With
well-fixed fecal smears, the staining characteristics of organisms
are reasonably consistent, and the color contrast between organisms
and background material render the organisms more easily visible
than in hematoxalin-stained smears. The cytoplasm of organisms will
usually stain a blue-green to purple, whereas nuclear chromatin,
chromatoid bodies and other inclusions will stain red to reddish
purple.
[0077] Suitable specimens include human stool samples in SAF
preservative that have been concentrated with PVA added. The sample
can be applied to labeled microscope slides and allowed to air dry
(as described above). By way of illustration, we provide the
following protocol, which can be carried out with a Jung
Autostainer.RTM. instrument. The reagents required include alcohol
(70% and 95%), acid alcohol (90%), and iodine alcohol (70%). Once
the Jung Autostainer.RTM. has been turned on and self-initializes,
it is ready to be loaded with the reagents and slides. The staining
proceeds automatically as follows: (1) 70% iodine alcohol for 5
minutes; (2) 70% alcohol for 2-3 minutes; (3) 70% alcohol for 2-3
minutes; (4) trichrome stain for 10 minutes; (5) 90% acid alcohol
dip; (6) 95% alcohol dip; (7) 95% alcohol dip; (8) 95% alcohol for
five minutes; (9) 95% alcohol for five minutes; (10) 95% alcohol
for five minutes; and (11) hemo-de solution for 25 minutes. The
slides should not be allowed to dry out before a coverslip is
applied. The coverslip can be a liquid coverslip such as
Flo-Texx.RTM..
[0078] In connection with these preparatory and staining methods,
one can also consult: American Society of Clinical Pathologists,
Laboratory Diagnostics of Intestinal Parasite Infection, CDC
training course material; Wheatley, Am. J. Clin. Pathol.
21:330-991; Ash and Orihel, supra; NCCLS, Procedures for the
Recovery and Identification of Parasites from the Intestinal Tract.
M28-A Vol. 17 No. 23, 1997; and Jung Autostainer.RTM. Instruction
Manual.
[0079] Upon inspection under the microscope, the cytoplasm of
protozoan trophozoites should be a blue-green color and may be
tinged with purple. Cysts tend to be more purple. Nuclei and
inclusions (chromatoid bars, RBCs, and bacteria) and Charcot-Leyden
crystals have a red color and are sometimes tinged with purple.
Glycogen is dissolved by the fixatives and appears as a clear area
in the organism. The background material usually stains green,
which provides strong contrast with the protozoa. As noted, the
contrast is often sharper than that observed with hematoxylin
staining. Helminth ova, Balantidium coli, Entamoeba coli cysts and
Isospora belli oocysts are best seen in wet preps, and acid-fast
stains are recommended for Cryptosporidium.
[0080] The protozoa, host cells, yeast cells, and any other
artifacts can be counted if desired and classified as rare, few,
moderate, or many. The Table below provides recommended limits for
these categories.
TABLE-US-00001 Wet Prep (concentration) Enumeration Protozoa (#/20
40X fields) Helminth (#/22x40 coverslip) Rare <4 <4 Few 4-6
4-6 Moderate 6-20 6-20 Many >20 >20
[0081] For yeast, 1-3 per hpf constitutes a rare number; 4-5 per
high powered fielf constitutes few; 6-10 per hpf constitutes a
moderate number; and >10 per hpf constitutes many.
[0082] Other parasites that can be assessed in the context of the
present methods include Giardia and Cryptosporidium. These
organisms can be detected in a variety of ways, including enzyme
immunoassay (EIA) for specific antigens (e.g., GSA 65 and CSA)
present in aqueous extracts of human stool samples. More
specifically, one can employ the ProspecT.RTM. microplate assay
according to the manufacturer's instructions to simultaneously
detect GSA 65 and CSA. Briefly, stool specimens are added to
break-away microplate wells to which anti-GSA 65 and anti-CSA
antibody are bound. The antibodies are conjugated to an enzyme that
later produces a detectable product when bound by an antigen. After
unbound material is washed away, an enzyme substrate (TMB where the
enzyme is horseradish peroxidase) is added to the well. In a
positive reaction, the development of color indicates that
parasitic antigens are present in the sample. In a negative
reaction, antigen is either absent or present in insufficient
amounts to be detected, and no colored reaction product develops.
Acceptable samples include unpreserved stool, stool samples in
Cary-Blair medium, SAF medium, or 10% formalin. Ideally, fresh
samples have been frozen, samples in Cary-Blair medium are
refrigerated (2-8.degree. C.) and tested within one week of
collection, and samples in SAF, 10% formalin or MF transport media
are refrigerated or stored at room temperature and tested within
two months. Stool samples treated with PVA are not acceptable for
testing.
[0083] Similar assays can be configured to detect other antigens so
long as an antigen-specific binding protein (e.g., an antibody) is
available. For example, the ProspecT.RTM. Entamoeba
histolytica/dispar microplate assay is available for the detection
of EHSA (E. histolytica specific antigen).
[0084] In assays such as the ProspecT.RTM. assay, acceptable
samples include unpreserved stool, stool samples in Cary-Blair
medium, SAF medium, or 10% formalin.
[0085] Any of the methods described herein can include a step of
identifying a patient who is a candidate for evaluation. In some
cases, a physician or other healthcare provider may suspect
giardiasis, caused by Giardia lamblia, or cryptosporidiosis, which
is caused by Cryptosporidium sp. G. lamblia are transmitted via
ingestion of viable cysts, and this occurs more frequently among
children and in groups of people who live in close quarters. The
acute stage occurs about 12-20 days after ingestion and typically
lasts only a few days. The patient may complain of diarrhea and
flatulence without the presence of blood in the stool or other
hallmarks of inflammatory bowel disease. Malaise, malabsorption,
anorexia, abdominal cramping, weight loss, and general weakness are
some of the other manifestations of infection. The disease can
linger for weeks or months as a chronic presentation.
[0086] Cryptosporidiosis is a serious intestinal disease that is
commonly observed in people who work with young children, animal
handlers, and travelers, and it is particularly dangerous to
individuals who are immunosuppressed or otherwise weakened or
compromised. Symptoms include diarrhea, abdominal pain, nausea and
vomiting, fever, malaise and respiratory problems, which may last
from several days to more than a month.
[0087] Any of the methods described herein can include a step in
which the results of one or more of the assays described herein are
correlated with patient symptoms and any other clinically relevant
information.
EXAMPLES
Example 1
[0088] A 32 year-old male patient presents with abdominal
discomfort and alterations in bowel movements (3-4 times/month)
since returning from traveling in Nepal six months earlier. He has
been evaluated for parasites at a local hospital. An EIA for
Entamoeba histolytica was negative. He has difficulty when eating
sweets or other sugar-rich foods, which leads to an urge to use the
bathroom. The patient presented to a doctor who suspected Irritable
Bowel Syndrome. While the doctor was concerned about the potential
underlying causes of pain and discomfort, hospitalization and
colonoscopy were not considered necessary at that time. The doctor
elected to perform stool testing to evaluate digestion,
inflammation, infection, and gut microflora. The following results
were noted: [0089] Pancreatic Elastase=188 (nl>200) [0090]
Calprotectin=22 (nl<50) [0091] Eosinophilic Protein X=8.9
(nl<7.0) [0092] Microscopy--positive for Blastocystis
hominis
[0093] The patient was prescribed pancreatic enzymes with meals,
treated with a prescription anti-parasitic agent, and offered
L-glutamine to support healing of the gastrointestinal lining.
Within one week, the patient began to experience improvement in
symptoms he had experienced for the previous six months.
L-Glutamine was continued for six weeks, and pancreatic enzymes
were continued for three months, as were supplemental
probiotics.
[0094] After three months, the symptoms had completely resolved and
medications could be ceased. The patient remains symptom-free more
than one year from the initial visit.
Example 2
[0095] A 42 year old female presents with a three year history of
irregular bowel movements and intermittent lower abdominal pain,
for which she has seen her previous primary care provider many
times. She also has been diagnosed with depression and is currently
using an SSRI for depression. She notes that the tri-cyclic
anti-depressant she used previously did not significantly affect
her abdominal pain. She has used fiber, anti-diarrheals,
probiotics, and dietary changes in the past, to no avail.
[0096] Upon presentation the doctor chooses to run a stool test to
evaluate the root cause of this illness. While waiting for the
stool test to return, empiric use of probiotics at 10 billion cfu
per day has made no difference.
[0097] Upon testing, pancreatic elastase was determined to be 482
(nl>200). At this level, one would conclude that the patient has
normal pancreatic function and no need for pancreatic enzyme
support. Calprotectin was determined to be 286 (nl<50). This
level of elevation indicates severe gastrointestinal tract
inflammation and warrants referral to a specialist for further
investigation (i.e., colonoscopy). There was no evidence of
parasite infection or altered gut flora.
[0098] While no clinical intervention is indicated, the patient was
referred for colonoscopy for inflammatory changes in the
gastrointestinal tract. She is diagnosed with ulcerative colitis
and placed on steroids and 5-ASA. About 1-10% of patients with IBS
go on to develop IBD. The stool test, with its use of a stool-based
marker for GI inflammation, allowed for early detection and
appropriate referral of this patient to the gastroenterologist.
Subsequent evaluations of GI inflammation with the calprotectin
biomarker can be used to monitor mucosal healing and determine if
repair is sufficient or complete.
Example 3
[0099] A 21 year old female presents with a history of intermittent
recurrent abdominal cramping, bloating/gas and occasional diarrhea
over the past two years. She has been evaluated by her college
health clinic on numerous occasions, was told that her symptoms
were primarily `stress related,` and was advised to cut back on her
coffee intake during the day. She was recently evaluated for
complaints of fatigue, and was diagnosed with hypothyroidism
(including elevated thyroid antibodies) for which she is being
treated. While home during a break, the patient was asked to do a
stool analysis by her primary care doctor who was concerned that
some underlying etiology had not been determined. Results were
significant for the following: [0100] Pancreatic Elastase=150
(nl>200) [0101] At this level, the pancreatic elastase
clinically indicates moderate pancreatic insufficiency and need for
pancreatic enzyme support. Evaluation of the underlying cause of
pancreatic insufficiency is warranted. [0102] Calprotectin=22
(nl<50) [0103] A normal Calprotectin level indicates no
inflammation due to neutrophilic activity in the gastrointestinal
tract. [0104] Eosinophil Protein X=13.1 (nl<7.0) [0105] An
elevated Eosinophil Protein X may be associated clinically with
Celiac disease, parasitic infections, and/or IgE mediated food
allergies. [0106] Microscopy--no evidence of parasite infection or
altered gut flora.
[0107] Given that Celiac disease can be consistent with the
patient's clinical symptoms, and may be indicated on stool analysis
by an elevated Eosinophil Protein X and compromised pancreatic
exocrine function (low Pancreatic Elastase), the doctor decided to
evaluate the patient for Celiac disease. Blood serologies (positive
Tissue Transglutaminase IgA and positive anti-Endomysial antibody)
confirmed the diagnosis of Celiac disease. The patient was placed
on a gluten-free diet and given pancreatic digestive enzyme
support. At follow-up, the patient reported that her symptoms had
markedly improved--appearing only during times of dietary
indiscretion.
[0108] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *