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Abstract

Most abdominal disorders present with a limited number of overlapping symptoms. Blood tests are not routinely available for use in diagnosis and so investigation tends to require complex imaging procedures or endoscopy and biopsy. These are invasive for the patient, may be associated with morbidity and mortality and have considerable resource implications. Biochemical tests on a single sample of faeces are therefore a valuable alternative. Measurement of faecal calprotectin has been shown to have a role in the diagnosis of inflammatory bowel disease and in its monitoring. Lactoferrin is also of benefit used in this way. Faecal elastase has been demonstrated to be of use in the diagnosis of pancreatic insufficiency. A number of faecal markers have been explored in colorectal cancer. Faecal occult blood testing is used for population screening, but the metabolomic marker tumour, M2-pyruvate kinase, has potential for use in both diagnosis and screening. DNA testing has advantages in colorectal cancer but the exact applications of such tests require further evaluation.

Introduction

Gastrointestinal disorders are common in clinical practice. Common organic causes include infection, inflammatory bowel disease (IBD) and neoplasia. Their symptoms overlap each other and those of functional disorders (clinical syndromes defined by symptoms without identifiable cause), making differential diagnosis difficult. Blood tests are often non-specific and diagnosis may require complex imaging or endoscopic procedures. These procedures are invasive, expensive and associated with morbidity and mortality. Attention has therefore turned to investigation of faeces, looking for markers that can be used in screening, diagnosis and follow-up of gastrointestinal disease. This review will describe some of the newer faecal tests available for investigation of gastrointestinal disease.

Inflammatory bowel disease

IBD is a group of inflammatory conditions of the small intestine and colon consisting principally of Crohn's disease and ulcerative colitis. The aetiology of IBD is unknown but is likely to involve genetic predisposition together with environmental factors and immune dysfunction.

The incidence of Crohn's disease is approximately 0.5–24.5/100,000 and that of ulcerative colitis 0.1–16/100,000 inhabitants/year with prevalence rates of IBD of up to 396/100,000 inhabitants.¹

Symptoms of IBD are determined by the anatomical location and the degree of inflammation but can include abdominal pain, diarrhoea and rectal bleeding.

Differentiation between Crohn's disease and ulcerative colitis can be made on the basis of histology. Crohn's disease is characterized by granulomatous inflammation which is transmural and can affect any part of the gut, although most commonly the ileum and colon, with a tendency to fistula formation. In ulcerative colitis, inflammation is confined to the mucosa of the rectum and colon and is characterized by ulceration, crypt abcesses and goblet-cell depletion. Treatment of IBD requires active management with suppression of the immune response.

Laboratory tests used in the diagnosis and monitoring of IBD may include measurement of erythrocyte sedimentation rate (ESR), plasma viscosity, C-reactive protein (CRP), albumin and platelet count. However, none of these is specific for gastrointestinal inflammation. In both Crohn's disease and ulcerative colitis, there is infiltration of leukocytes into the gut wall and subsequent loss of both leukocytes and their products into the bowel lumen and hence the faeces.

High faecal neutrophil counts are observed in active IBD,^2,3 but lack applicability as a diagnostic test due to the limited lifespan of the cells. Faecal excretion of indium-111-labelled leukocytes is a recognized diagnostic test for gastrointestinal inflammation. It has been shown to correlate with endoscopic and histological findings and to have a sensitivity of 97% for the diagnosis of IBD,⁴ but requires the patient to be exposed to radiation and to perform a prolonged faecal collection. Candidate faecal markers that can be measured in a single sample have therefore been sought. Suggested markers have included myeloperoxidase, lysozyme, polymorphonuclear elastase (PMN-E),⁵ calprotectin and lactoferrin, the latter two having been the most widely studied. Myeloperoxidase is increased in faeces in IBD but its short half-life limits its usefulness.¹ Studies involving lysozyme have reported it to have relatively poor stability⁶ and a limited value in small bowel disease.¹ PMN-E also has limited stability.⁶

Calprotectin and lactoferrin

Calprotectin, previously termed L1 protein, is a calcium and zinc binding protein with a molecular weight of 36 kDa, which is a member of the S100 family. It is derived predominantly from neutrophils and constitutes about 60% of their cytosolic proteins.⁷ It is found in various body fluids in proportion to the severity of any existing inflammation. Calprotectin has bacteriostatic and fungistatic properties and is also able to inhibit fibroblastic cell growth and to induce apoptosis in various host cells.⁸

Lactoferrin is a 76-kDa member of the transferrin family of iron-binding glycoproteins.⁹ It is found in many human body fluids including serum, milk, synovial fluid and tears and stored in secondary granules of polymorphonuclear granules. Lactoferrin was originally viewed purely as an iron-binding protein in milk but it is now clear that it has direct antimicrobial activities against some bacteria, viruses and fungi and may also enhance phagocytic activity of neutrophils via opsonic and neutrophil-stimulating mechanisms.¹⁰

Stability

Calprotectin is stated by many as being stable for up to one week at room temperature.^5,11,12 However, they quote a single study as their source.¹³ In this study, it is of note that there was a significant decrease of 17.5% after 48 h storage at room temperature and while there was no difference in the mean calprotectin concentration after seven days of storage at 20°C, individual samples showed marked variation. Lactoferrin has been shown to be more stable than calprotectin after storage at more than 48 h at room temperature¹⁴ and may be a better option if delay in transit to the laboratory is anticipated.

Sample requirements

Measurement of calprotectin in single samples has been shown to have an excellent correlation with 24-h collections,¹³ making a random sample appropriate for clinical use. Non-steroidal anti-inflammatory drugs, such as indomethacin and aspirin, can increase faecal calprotectin and lactoferrin, to an amount consistent with that found in IBD, probably due to an associated enteropathy.^15,16 Although patients with menstrual and nasal bleeding have been excluded from studies to avoid potential contamination of faecal samples by neutrophils in blood, it has been estimated that a bleeding volume of 100 mL is required to cause an elevated calprotectin.¹⁷

Enzyme-linked immunosorbent assay

Early studies of calprotectin used an enzyme-linked immunosorbent assay (ELISA) involving a rabbit anti-calprotectin antibody. Results were expressed per litre of faecal homogenate.¹³ A polyclonal antibody has been available since 2000 that recognizes six epitopes of calprotectin and is the basis of the commercial assays that are now available (e.g. Phical, Calpro AS, Lysaker, Norway and Buhlmann Laboratories AG, Basel, Switzerland). These report results in μg/g of faeces. Good correlation has been demonstrated between the two.¹⁸ For the older assay, a cut-off of 10 mg/L was recommended, which corresponds to the manufacturer's recommended cut-off of 50 μg/g for the polyclonal assays. The reference range for calprotectin has been shown to be up to 10 times higher in infants^19,20 and an upper limit of 166 μg/g has been proposed for children of up to nine years.²¹ Among adults, an increase in faecal calprotectin concentration has been demonstrated with increasing age.^21,22 While 50 μg/g is proposed for screening purposes, different cut-off values are suggested for different patient groups. When used to diagnose IBD, cut-offs of 100²³ and 150 μg/g²⁴ have been used.

The manufacturer's reference range for lactoferrin is <7.25 μg/g. In infants, lactoferrin may be present in faeces in very high concentrations, secondary to dietary intake of milk protein and a reference range of <29 μg/g has been proposed in children aged 2–9 y.²¹

Rapid tests

Various immunochromatographic tests are now available for measurement of calprotectin and lactoferrin, which enable rapid measurement of single samples as an alternative to ELISA. Some of these tests offer qualitative results, for example, IBDEZEVUE test for lactoferrin (TechLab, Blacksburg, VA, USA) or semi-quantitative results, for example, PreventIDCaldetect (Preventis GmbH, Bensheim, Germany) for calprotectin. Other immunochromatographic tests for calprotectin offer the ability of linkage to a scanner (e.g. CalproScan, Calpro AS) or use of a dedicated platform (e.g. Quantum Blue, Buhlmann AG) to provide a quantitative result.

Studies comparing the rapid tests with the standard ELISA have shown them to perform well (Table 1).

Table 1

Evaluation of rapid tests of inflammatory markers for the diagnosis of inflammatory bowel disease

Test	Cut-off (μg/g)	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
Prevent	15	100	95	82	100
IDCalDetect²⁵	60	61	98	88	91
Calprotectin ELISA	50	96	87	65	99
PreventIDCalDetect²⁶	15	96	70	79	94
	60	66	100	100	72
Calproscan²⁷	50	93	86	99	99
PreventIDCalDetect	60	67	95	67	99
IBDEZEVUE	n/a	67	97	73	99
Prevista Calprotectin²⁸	50	89	80	59	96
Calprotectin ELISA	50	100	79	60	100

PPV, positive predictive value; NPV, negative predictive value; ELISA, enzyme-linked immunosorbent assay

The CalDetect offers results that are <15 μg/g, between 15 and 60 μg/g and >60 μg/g. A recent study concluded that the rapid test was useful as an exclusion test using the 15 μg/g cut-off but recommended that the ELISA should be used for monitoring IBD activity and for accurate estimation of concentrations >50 μg/g.²⁶ The authors suggested matrix effects with aqueous and very small samples as a cause of the inaccuracy. Thus, immunochromatographic tests are likely to be useful to enable rapid turnaround of results in certain clinical settings where the negative predictive value of the test is of particular value but may not be suitable for monitoring of patients with known IBD where a high concentration is likely.

Diagnosis of IBD

Faecal calprotectin and lactoferrin tend to be significantly elevated in patients with IBD compared with healthy subjects, but for clinical purposes, it is their use as markers of IBD in patients presenting with similar symptoms that is relevant, particularly those presenting with irritable bowel syndrome (IBS). Various studies have been performed to evaluate the use of calprotectin and lactoferrin in the diagnosis of IBD (Table 2).

Table 2

Studies of diagnostic use of (a) calprotectin and (b) lactoferrin in inflammatory bowel disease

	Patients
Study	(n) age	Disease	Sensitivity (%)	Specificity (%)	Cut-off μ(g/g)
(a) Calprotectin
Tibble et al. ²⁴	31 adults	CD	100	97	150
Carroccio et al. ²⁹	8 children	CD	100	96	50
	10 adults	CD	100	80	50
Tibble et al. ²⁴	116 adults	CD	98	86	50
Fagerberg et al. ³⁰	22 children	IBD	95	93	50
Bremner et al. ³¹	43 children	IBD	95	72	50
Limburg et al. ³²	29 adults	IBD	94	83	100
Schroder et al. ³³	25 adults	CD	93	100	15
	20 adults	UC
Canani et al. ³⁴	17 children	CD	93	89	100
	10 children	UC
Thjodleifsson et al. ³⁵	49 adults	CD	88	91	50
Otten et al. ²⁵	23 adults	IBD	87	90	50
Costa et al. ³⁶	49 adults	CD	81	82	50
	82 adults	UC
D'Inca et al. ³⁷	31 adults	CD	79	74	80
	46 adults	UC
Summerton et al. ³⁸	4 adults	CD	79	–	50
	10 adults	UC
Bunn et al. ³⁹	21 children	CD	65	100	50
	16 children	UC
Kaiser et al. ⁴⁰	32 adults	CD	63	86	50
	27 adults	UC
Silberer et al. ⁴¹	39 adults	IBD	61	–	18.6
(b) Lactoferrin
Dai et al. ¹⁴	13	IBD	92	80	240
Schoepfer et al. ⁴²	28	UC	91	96	7
Fine et al. ⁴³	103	IBD	90	98	–
Langhorst et al. ⁴⁴	42	UC	90	88	324
Walker et al. ⁴⁵	106	IBD	87	68	7.25
Schoepfer et al. ⁴⁶	36	IBD	86	100	50
	36	CD	83	96
Schroder et al. ³³	45	IBD	82	100	7.3
Langhorst et al. ⁴⁴	43	CD	82	60	7.25
Otten et al. ²⁶	23	IBD	78	90	7.25
Kane et al. ⁴⁷	215	IBD	78	90	4
D'Inca et al. ³⁷	77	IBD	76	67	–
Saitoh et al. ⁴⁸	18	UC	67	–	–
Hirata et al. ⁴⁹	40	CD	63	–	6.5
Saitoh et al. ⁴⁸	13	CD	54	–	–
Hirata et al. ⁴⁹	62		47	–	6.5

CD, Crohn's disease; IBD, inflammatory bowel disease; UC, ulcerative colitis

From these data, using statistical techniques, mean sensitivities of 80% for calprotectin and lactoferrin and mean specificities of 76% for calprotectin and 82% for lactoferrin have been calculated for the diagnosis of IBD.^12,50

There is therefore potential for faecal tests to be used as a first-line test, with endoscopy only being used when the probability of organic disease is relatively high.

A recent meta-analysis concluded that in the investigation of suspected IBD in adults, screening by measurement of faecal calprotectin would result in a 67% reduction in the number of patients requiring endoscopy.⁵¹ A recent NHS evaluation by the Centre for Evidence-based Purchasing used both clinical and economic data to evaluate the effectiveness of calprotectin and concluded that it would be less costly and more accurate than the measurement of CRP or ESR in the differentiation of IBD from IBS in primary care.⁵² However, there is not yet sufficient evidence to support the use of the test in this setting.⁵³

Assessment of IBD activity

IBD is an inflammatory condition which can be treated with therapies targeted against the inflammation. However, some features of the disease, for example, diarrhoea secondary to bile salt malabsorption or abdominal pain relating to a fibrous stricture, may be the result of non-inflammatory processes or complications. Clinical scoring systems (for example, the Harvey Bradshaw and Crohn's disease activity indices) have been developed but involve an element of subjectivity which can be influenced by non-inflammatory components; their correlation with endoscopic and histological assessment of disease activity is suboptimal.⁵⁴ A relationship between IBD activity, evaluated by clinical, endoscopic and histological means, has been noted for calprotectin¹² and lactoferrin.⁵⁰ Calprotectin has been noted to correlate better with histological than endoscopic findings in assessing disease activity in IBD.⁵⁵

Lactoferrin has been shown to correlate with histological inflammation in Crohn's disease and endoscopic and histological inflammation in ulcerative colitis.³⁷ The tests are probably equally useful in assessing IBD activity,⁵⁶ but their exact role for this purpose requires further evaluation.

Prediction of relapse

IBD is characterized by relapses and remissions. The ability to predict relapse would allow appropriate intensification of treatment for those affected and possible reduction in maintenance therapy for others.

Calprotectin has been shown to have a sensitivity of 90% and a specificity of 83% for prediction of relapse in IBD at a cut-off of 50 μg/g.⁵⁷ A subsequent study suggested a similar accuracy in prediction of relapse in ulcerative colitis (sensitivity 89%, specificity 82%) but a specificity of only 43% in Crohn's disease.⁵⁸ Reasons for the discrepancy are unclear. A single measurement of lactoferrin showed a 100% sensitivity for prediction of relapse of IBD in a three-month period and a 62% sensitivity and 65% specificity over 12 months.⁵⁹

Intermittent measurement of faecal calprotectin and of lactoferrin in a clinical setting may be of use in order to determine early detection and prediction of relapse.¹ However, the time-course of elevation of faecal markers relative to clinical evidence of relapse is not yet fully determined, so the optimum frequency of measurement for this purpose is not known.

Assessment of response to treatment

Patients with IBD in whom gastrointestinal inflammation is not fully controlled have a higher risk of relapse. Confirmation of cure by endoscopy in all patients is not realistic, hence the ability to assess healing from a biomarker would be useful. Faecal calprotectin and lactoferrin have been shown to fall after infliximab therapy.^60,61 Calprotectin has been measured during glucocorticoid therapy and was found to fall in association with clinical activity.⁶² Failure of markers to fall on treatment may indicate treatment failure and an increase in marker concentration, even without clinical symptoms, may indicate a requirement for further therapy.

Assessment of response to surgery

Both calprotectin and lactoferrin have been found useful in the assessment of patients after bowel surgery, the persistence of elevation suggesting ongoing inflammation.⁶³ For some patients with ulcerative colitis, colectomy is performed together with ileal pouch-anal anastomosis. Subsequent symptoms may be due to inflammation (e.g. pouchitis) or non-inflammatory problems such as irritable pouch syndrome.

At a cut-off of 50 μg/g, calprotectin has been shown to distinguish pouchitis from a non-inflamed pouch with a sensitivity of 90% and a specificity of 76.5%.⁶⁴ In this circumstance, lactoferrin has been shown to have a sensitivity of 100% and a specificity of 85%⁶⁵ and has been further evaluated and found to be a cost-effective initial screen for the evaluation of symptomatic patients.⁶⁶

Organic bowel disease

More recently, attention has turned to whether calprotectin and lactoferrin could be of use in the differentiation of organic from functional bowel disease (Table 3).

Table 3

Studies of calprotectin to distinguish organic from functional disorders

	Patients
Study	n	Age	Organic disease	Sensitivity (%)	Specificity (%)	Cut-off (μg/g)
Bunn et al. ⁵⁵	22	Children	16	100	86	31.5
Dolwani et al. ⁶⁷	73	Adults	25	100	79	60
Fagerberg et al. ³⁰	36	Children	22	95	93	50
Jeffery et al. ⁶⁸	105	Adults	14	93	92	50
Tibble et al. ⁶⁹	602	Adults	263	89	79	50
Sanchez et al. ⁷⁰	190	Adults	73	85	81	217
Limburg et al. ³²	110	Adults	29	83	83	100
Costa et al. ³⁶	239	Adults	157	83	82	50
				81	88	60
Chung-Faye et al. ⁷¹	131	Adults	88	80	74	25
D'Inca et al. ³⁷	67	Adults	–	78	88	50
Tibble et al. ⁷²	233	Adults	105	77	84	50
Carroccio et al. ²⁹	50	Children	35	70	93	50
	70	Adults	30	64	80

Using data from 14 of these studies, a mean sensitivity of 83% and a specificity of 84% were calculated for use of calprotectin in the diagnosis of organic intestinal disease.¹² Lactoferrin has been studied less than calprotectin in this context, but a recent study evaluating the efficacy of the two markers in detecting organic disease as assessed by colonoscopy found them to be equally commendable.³⁷

Some organic diseases are not characteristically associated with a neutrophil response, hence the use of calprotectin and lactoferrin in the diagnosis of organic bowel disease would be expected to be associated with a lower sensitivity than diagnosis of IBD.

In summary, calprotectin and lactoferrin are neutrophil proteins, which are markers of gastrointestinal inflammation. Both proteins have been shown to be sensitive and specific faecal tests for the diagnosis of IBD and both have the potential for use in a number of ways in the management of the condition. IBD is not the only cause of a raised concentration of faecal calprotectin or lactoferrin; other causes include blood loss, use of non-steroidal anti-inflammatory drugs, gastrointestinal infection and neoplasia. The markers may therefore have a role in the differential diagnosis of organic from functional bowel disease and in screening out cases of IBS. The finding of a negative calprotectin or lactoferrin result in a low-risk patient may allow them to be discharged without further invasive investigation.

Pancreatitis

Chronic pancreatitis is an inflammatory disease causing structural changes in the pancreas, which ultimately lead to impairment of exocrine and endocrine pancreatic function.⁷³ Patients may present with pain, classically in the epigastrium radiating to the back, although about 20% of those affected present with endocrine or exocrine pancreatic dysfunction in the absence of pain.⁷⁴ Exocrine pancreatic dysfunction leads to diarrhoea, steatorrhoea and weight loss. The annual incidence of chronic pancreatitis is estimated to be 3–9 cases/100,000,⁷⁵ with alcohol involved in the aetiology of about two-thirds. Although chronic pancreatitis is defined by the presence of histological damage to the pancreas, biopsy specimens are difficult to obtain, which precludes this approach to diagnosis. Instead, diagnosis is usually based on detection of abnormal structure or function.

Structural tests for chronic pancreatitis include imaging investigations such as computed tomography, magnetic resonance cholangio-pancreatography (MRCP), endoscopic retrograde cholangiopancreatography and endoscopic ultrasound.

A diagnosis of chronic pancreatitis can also be made by assessing pancreatic function. Such tests can be divided into invasive tube pancreatic function tests and relatively simple biochemical tests. The object of the tube tests is to collect and measure a component of pancreatic secretions in order to determine secretory capacity after stimulation by infusion of secretagogues; examples include the traditional secretin and cholecystokinin (CCK) stimulation tests.⁷⁶ Caerulin and bombesin can also be used in place of CCK. More recently, enhanced imaging function tests such as the secretin-enhanced MRCP have been developed.⁷⁷

Simple biochemical tests for chronic pancreatitis are designed to avoid invasive procedures and to be performed relatively easily on an outpatient basis.

Faecal fat

The measurement of fat in a 72-h faecalt collection used to be a routine test in the evaluation of pancreatic function. However, concerns about its clinical appropriateness and analytical performance have caused its position to be reconsidered.⁷⁸ Current guidelines no longer propose it as an investigation for pancreatic insufficiency, although do suggest that in situations where malabsorption is suspected and investigations of the cause have proved negative, a single stool for faecal fat assessment by a method such as Sudan staining or steatocrit may be appropriate.⁷⁹

Faecal elastase

The pancreolauryl test became established as a diagnostic test for pancreatic exocrine dysfunction. Its sensitivity for detecting severe insufficiency was 85%, although a lesser figure of 39% has been quoted for mild/moderate disease.⁷⁹ The fluorescein dilaurate capsules required were withdrawn from sale in 2005 due to manufacturing and supply issues, requiring another biochemical test for pancreatic insufficiency. For most laboratories this is now faecal elastase.

Pancreatic elastase-1 is a pancreas-specific protease that passes unchanged through the intestine and whose concentration in faeces is six times that in duodenal juice.⁸⁰ There are two commercially available tests: one uses a monoclonal (ScheBo Biotech, Giessen, Germany) and the other a polyclonal (Bioserv Diagnostics, Rostock, Germany) antibody system utilizing four different antibodies. Both tests are ELISAs with a total incubation time of 3.5 h. The assays share independently established but equivalent reference ranges of >200 μg/g faeces for normal pancreatic function, 100–200 μg/g faeces for moderate insufficiency and <100 μg/g faeces for severe pancreatic insufficiency.⁸¹ The faecal elastase assay does not cross-react with exogenous porcine pancreatic enzymes used in supplementation, so patients can remain on therapy during testing if required.⁷⁷ Where the performance of the assays have been directly compared, some studies have shown that the polyclonal ELISA may have a similar specificity but slightly greater diagnostic sensitivity than the monoclonal.^82,83 However, a recent, large study concluded that the assays were equivalent in their ability to classify patients as normal or as moderately or severely pancreatic insufficient.⁸¹

A number of studies have looked at the sensitivity and specificity of faecal elastase in the diagnosis of pancreatic insufficiency. The results are summarized in Table 4; the studies themselves are not directly comparable due to differences in the protocols used.

Table 4

Studies on the clinical value of faecal elastase

Study	Patients (n)	Diagnosis	Sensitivity (%)	Specificity (%)	Categorization
Loser et al. ⁸⁴	79	SCT	100		Severe pancreatic insufficiency
			100		Moderate pancreatic insufficiency
			63		Mild pancreatic insufficiency
				93	Normal pancreatic function
Luth et al. ⁸⁵	127	SCT	100		Severe pancreatic insufficiency
			86		Moderate pancreatic insufficiency
			65		Mild pancreatic insufficiency
				57	Normal pancreatic function
Gullo et al. ⁸⁶	140	ERCP, ultrasound	100		Severe chronic pancreatitis
			76.9		Moderate chronic pancreatitis
			22.2		Mild chronic pancreatitis
				95.8	Healthy controls
Amann et al. ⁸⁷	36	SPT,	100		Severe chronic pancreatitis
		X-ray,	43		Mild/moderate pancreatitis
		ERCP		29	Healthy controls
Dominguez-Munoz et al. ⁸⁸	69	ERCP, CT	100		Severe/moderate pancreatitis
			0		Mild chronic pancreatitis
				83	Non-pancreatic disease
Stein et al. ⁸⁹	29	SPT	96		Severe pancreatic insufficiency
			88		Moderate pancreatic insufficiency
				94	Mild pancreatic insufficiency
Lankisch et al. ⁹⁰	64	SPT	82		Severe pancreatic insufficiency
			33		Moderate pancreatic insufficiency
			40		Mild pancreatic insufficiency
				94	Regular pancreatic function
Hardt et al. ⁹¹	213	ERCP	76.5		Severe chronic pancreatitis
				86	Moderate/severe chronic pancreatitis
Naruse et al. ⁹²	117	SST	72.7*		Severe pancreatic insufficiency
			12.5		Moderate pancreatic insufficiency
			16.5		Mild pancreatic insufficiency

ERCP, endoscopic retrograde cholangiopancreatography; SPT, secretin-pancreozymin test; SCT, secretin-caerulin test; SST, secretin stimulation test; CT, computed tomography

All studies used the monoclonal assay except those marked with *

It appears, therefore, that measurement of faecal elastase is an excellent test for the detection of severe pancreatic exocrine dysfunction, although it is less sensitive in mild and moderate disease. It has become established as an investigation of pancreatic exocrine function in those patients presenting with diarrhoea of unknown aetiology. A limitation of the faecal elastase test is potential variability due to stool water content, with the possibility of false-positive results due to a dilutional effect in watery samples.⁹³ A dry faecal extraction procedure has been described, which overcomes water interference and enables samples to be used which would otherwise be considered unsuitable for analysis.⁹³

A further use of faecal elastase is in the investigation of patients with cystic fibrosis, in order to categorize their pancreatic status. Exocrine pancreatic insufficiency is one of the major clinical features of cystic fibrosis. Pancreatic function tends to decline early in life, but some patients may remain pancreatic sufficient until later in life. The measurement of faecal elastase has been shown to be a suitable test both for classification of pancreatic function in patients at diagnosis of cystic fibrosis⁹⁴ and for longitudinal follow-up of their exocrine pancreatic function.⁹⁵

Colorectal carcinoma

Colorectal carcinoma is the second most common cancer with respect to incidence and mortality in England and Wales, causing about 15,000 deaths annually.⁹⁶ The probability of developing colorectal cancer increases with age. Therefore, the incidence is rising since the number of elderly individuals, as a proportion of the total population, is rising.

Most colorectal cancers develop from malignant change in adenomas that have been present for some considerable time, giving rise to the possibility of screening to detect them at an early or premalignant stage. Colonoscopy is the ‘gold standard’ for the detection of colorectal cancers, but when implemented as a screening procedure is, perhaps unsurprisingly, not well received by the public. For example, take-up in Germany is only 1.7%.⁹⁷ Non-invasive faecal screening tests are much more acceptable to patients as they are less invasive and safer.

Faecal occult blood testing

Screening for colorectal cancer in asymptomatic individuals using faecal occult blood testing (FOBT) has been shown to be successful in reducing mortality from the disease.⁹⁸ Commonly used FOBTs (e.g. Haema-Screen, Immunostics, Ocean, NJ, USA) are guaiac-based and rely on the peroxidase-like activity of haem in faeces. Their sensitivity is reported as 95% and specificity as 15–30%.⁹⁹ Potential interferences include plant peroxidases in certain fruits and vegetables, dietary haem in red meat and sources of bleeding other than from colorectal cancer. However, their actual significance is debated¹⁰⁰ and advising dietary restriction prior to the test may affect patient compliance without reducing the positivity rate.¹⁰¹ Newer faecal immunochemical occult blood tests, sometimes abbreviated to FITs (e.g. Hema-select SPECIFIC, Immunostics) detect the globulin portion of human haemoglobin and hence are not subject to the same interferences, which may increase sensitivity and specificity.

In England, there is a Bowel Cancer Screening Programme in place using guaiac-based FOBT. In Scotland, a two-tier reflex FOBT/FIT algorithm is used. Interested readers are referred to National Guidance for more details.^102,103

A number of other faecal markers have been considered as candidates for screening tests for colorectal cancer, for example, albumin, lactoferrin, lysozyme and calprotectin but these lack specificity and sensitivity.¹⁰⁴ The metabolomic marker tumour, M2-pyruvate kinase (tumour M2-PK), shows promise as a screening test.

Tumour M2-pyruvate kinase

The metabolism of tumour cells is altered such that there is an increase in glycolytic metabolism with diversion of glycolytic products from energy production to synthetic processes. Pyruvate kinase (PK) is an important regulatory enzyme in this process. Several isoforms of PK are expressed in specific tissues and exist as tetramers. In tumour cells, including colorectal cancer, the isoenzyme M2-PK is preferentially expressed as a dimer (tumour M2-PK). Tumour M2-PK can be measured in faeces, which has led to interest in its potential as a tumour marker for colorectal cancer.

There have been a number of retrospective and prospective studies examining the use of tumour M2-PK in colorectal cancer screening (Table 5).

Table 5

The use of tumour M2-PK as a screening tool for colorectal carcinoma (CRC)

		Sensitivity (%)
Reference	Patients (n)	CRC	Adenoma	Specificity (%)
Koss et al. ¹⁰⁵	55	91	20, 60 (>1 cm)	92 (general population)
Koss et al. ¹⁰⁶	45	87.5	37.5, 60 (>1 cm)	100 (endoscopy controlled)
Naumann et al. ¹⁰⁷	232	85.2	45.8, 61.5 (>1 cm)	65.3 (endoscopy controlled)
Mulder et al. ¹⁰⁸	181	85	28	90 (endoscopy controlled)
Haug et al. ¹⁰⁹	982	85		79 (general population)
Shastri et al. ¹¹⁰	317	81.8	25.8, 20 (>1 cm)	71.1 (endoscopy controlled)
Tonus et al. ¹¹¹	96	77.8		92.9 (endoscopy controlled)
Vogel et al. ¹¹²	138	77.3	47.6	71.9 (endoscopy controlled)
Hardt et al. ¹¹³	204	73.3		78.8 (endoscopy controlled)
Hardt et al. ¹¹⁴	78	68.8	50	98 (general population)

These studies have shown tumour M2-PK to have an overall sensitivity of between 68.8% and 91% and an overall specificity of 71.9% and 100%.¹¹⁵

Some of the studies have looked at the performance of tumour M2-PK in patients with adenomas, sensitivity being between 20% and 61.5%, according to the size of the adenoma. There is also a correlation between tumour M2-PK concentration and tumour staging for both TNM (tumour, node, metastases) and Dukes’ classification and successful surgical intervention has been associated with reduction in tumour M2-PK.^105,113 Tumour M2-PK has therefore been recommended as a screening test for colorectal cancer in the general population.^115,116 It could also have a role in assisting prioritization of endoscopy and in screening between regular scheduled endoscopies undertaken in those at high risk of bowel cancer.

Genetic markers

A number of DNA mutations have been associated with colorectal cancer, making the examination of faeces for DNA studies of potential use for screening. DNA is stable in faeces and, unlike blood, is shed continuously from malignant lesions. Other advantages over FOBT include lack of the requirement for pretest preparation and avoidance of dietary and medicinal substances. Mutations tend to cause tumours over many years; hence screening is likely to be required less frequently than for other tests, although practical information about timescale is not readily available.

Colorectal cancers and adenomas arise from at least three genetic pathways – chromosomal instability, microsatellite instability and DNA methylation.¹¹⁷

Chromosomal instability is a feature of most human tumours and is characterized by allelic imbalance due to loss or gain of distinct chromosomal regions.¹¹⁸ It accounts for about 85% of sporadic colorectal cancer and almost all cancers in the familial syndrome adenomatous polyposis coli. Such tumours are characterized by the progressive accumulation of mutations and changes in key genes, which include the tumour suppressor genes adenomatous polyposis coli (APC), located on chromosome 5, and k-ras and the oncogene p-53 located on chromosome 17q.¹¹⁹ Mutations in APC tend to occur early in the development of adenomas and are followed by changes in the k-ras. Those in p-53 tend to be associated with a later stage of the neoplastic process and are found in large adenomas with severe dysplastic change.

Mismatch repair genes produce proteins that repair errors which occur during DNA replication. Loss of function of these genes can lead to the accumulation of errors within a DNA sequence.¹²⁰

Microsatellites are short, repeating sequences of DNA, which tend to accumulate errors in the presence of an impaired mismatch repair mechanism. This can lead to a loss of gene function. If the affected gene regulates cell growth then cancer may result. The microsatellite instability marker Bat-26 is a single locus of 26 consecutive adenine nucleotides relevant to colorectal cancer, particularly right-sided lesions.¹²¹

Hypermethylation of cytosine residues in CpG-rich sequences in the promoter regions of genes regulating cell growth have a role in malignant transformation of colorectal adenomas, for example, by down-regulating tumour suppressor genes.¹²² The first faecal DNA studies investigated single markers. They were successful in demonstrating that the same mutation that occurred in cancer tissue could also be detected in faeces, but sensitivity for detection of colorectal cancer tended to be less than 40%.¹²³ Different genetic pathways for the development of colorectal cancers are not necessarily mutually exclusive and can be combined in a single screening test.

This led to the development of commercial multimarker panels such as the PreGen-Plus (Exact Sciences Corporation, Marlborough, MA, USA), which involved a panel of 21 specific point mutations (10 in APC, 3 in k-ras and 8 in p-53, plus BAT-56 in addition to long DNA, a marker of disordered colonocyte apoptosis) (Table 6). A newer assay, the PreGen-Plus version 2, incorporates improvements in DNA extraction and stabilization and is associated with better sensitivity and specificity. Other developments have included studies looking at hypermethylation of the vimentin and secreted frizzled-related protein (SFRP) genes (Table 6).

Table 6

The use of faecal DNA as a screening tool for colorectal carcinoma

		Sensitivity (%)
Study	Test	Cancer	Adenoma	Specificity (%)
Ahlquist et al. ¹²⁴	PreGen-Plus	91	82	100
Brand et al. ¹²⁵	PreGen-Plus	69	–	–
Tagore et al. ¹²⁶	PreGen-Plus	63	57	98.2
Calistri et al. ¹²⁷	PreGen-Plus	62	–	97
Syngal et al. ¹²⁸	PreGen-Plus	62	27	–
Itzkowitz et al. ¹²⁹	PreGen-Plus version 2	83	86	82
Chen et al. ¹³⁰	Vimentin methylation	46	–	90
Huang et al. ¹³¹	SFRP2 methylation	94	52	96
Wang & Tang¹³²	SFRP2 methylation	87	62	93
Muller et al. ¹³³	SFRP2 methylation	77	–	77
Nagaska et al. ¹³⁴	SFRP2 methylation	63	32	91
Leung et al. ¹³⁵	SFRP2 methylation	30	12	87
Zhang et al. ¹³⁶	SFRP1 methylation	84	100	86

Faecal DNA testing has acceptable sensitivity and specificity as a screening test for colorectal cancer, but it remains expensive and the best marker or combination of markers is not yet determined. In addition, there is uncertainty over the optimal testing interval and how to manage patients with positive faecal DNA tests and normal colonoscopies. However, as faeces may contain DNA from any organ in communication with the gastrointestinal lumen, it is possible that the material could also be used to screen for malignancies in addition to those of the colon in the future.

Conclusions

Biochemical tests performed on faeces have been shown to be useful in several areas of gastroenterology. There is evidence that the measurement of calprotectin and lactoferrin have a role in the differentiation of IBD from IBS and in the monitoring of IBD and the prediction of relapse. The measurement of faecal elastase has been shown to be a sensitive and specific tool for the diagnosis of pancreatic insufficiency in patients with diarrhoea of unknown aetiology and also in those patients in whom malabsorption may be present. It is also of value in the assessment of pancreatic function in children with cystic fibrosis. A number of faecal markers have been explored in colorectal cancer, with FOBT currently in use for population screening. The use of faecal DNA testing has also been explored but its exact role requires further evaluation.

DECLARATIONS

Competing interests: None.

Funding: None.

Ethical approval: Not applicable.

Guarantor: RMA.

Contributorship: RMA.

Acknowledgements: None.

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New faecal tests in gastroenterology

Abstract

Introduction

Inflammatory bowel disease

Calprotectin and lactoferrin

Stability

Sample requirements

Enzyme-linked immunosorbent assay

Rapid tests

Diagnosis of IBD

Assessment of IBD activity

Prediction of relapse

Assessment of response to treatment

Assessment of response to surgery

Organic bowel disease

Pancreatitis

Faecal fat

Faecal elastase

Colorectal carcinoma

Faecal occult blood testing

Tumour M2-pyruvate kinase

Genetic markers

Conclusions

DECLARATIONS

References