Clinical value of duodenal biopsies – Beyond the diagnosis of coeliac disease

Pathology – Research and Practice 207 (2011) 538–544

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Pathology – Research and Practice journal homepage: www.elsevier.de/prp

Review Series of the Upper Gastrointestinal Tract

Clinical value of duodenal biopsies – Beyond the diagnosis of coeliac disease Marjorie M. Walker a,∗ , Nicholas J. Talley b,c a

Department of Histopathology, Imperial College London, St Mary’s Campus, London W2 1PG, UK Division of Gastroenterology and Hepatology, Davis Building E-6, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA c Faculty of Health, University of Newcastle, NSW, Australia b

a r t i c l e

i n f o

Keywords: Coeliac Disease Biopsy Histology Duodenum Intraepithelial lymphocytes

a b s t r a c t At upper gastrointestinal endoscopy to investigate unexplained diarrhea and iron deficiency anemia, duodenal biopsies are often taken to exclude a diagnosis of coeliac disease. While histology remains the gold standard for this diagnosis, recent developments in serological testing may overtake this as a first line test and biopsy restricted to confirming the diagnosis. Established coeliac disease on biopsy is straightforward, but early lesions may pose a challenge. Newer endoscopic procedures such as push–pull enteroscopy (balloon enteroscopy) with biopsy allow access to the small bowel beyond the second part of the duodenum. Controversy remains as to what constitutes the normal histology of the duodenum, and small bowel. Lymphocytic duodenosis (increased intraepithelial lymphocytes with normal villous architecture) in patients with negative coeliac serology can be associated with Helicobacter pylori, drugs, autoimmune and other diseases including food allergy. Full thickness small intestinal biopsies can aid in investigation of enteric neuropathies in severe dysmotility disorders. Biopsies are also taken to investigate malabsorption due to suspected infectious and metabolic disorders. Despite highly active anti-retroviral therapy (HAART), immunosuppressed patients may be affected by duodenal pathogens. The histology of duodenal mucosa in acid related disorders reflects the damage seen at endoscopy. Although the prevalence of duodenal ulcer disease is decreasing, drugs causing ulceration remain an important disease entity. Recent observations in functional bowel disorders suggest that the duodenum may be a key site for pathology. In functional dyspepsia, patients with early satiety may have excess eosinophil infiltration, and the mast cell is probably a key player in the irritable syndrome in the small intestine. © 2011 Elsevier GmbH. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coeliac disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis of coeliac disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biopsies – Where from and how many? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histological classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Architecture, villus crypt ratio and crypt hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intraepithelial lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How many is too many? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simplifying duodenal biopsy reporting of coeliac disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HLA typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lymphocytic duodenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malabsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small intestinal bacterial overgrowth (SIBO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disaccharidase deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mural neuromuscular disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opportunistic infections and HIV enteropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. E-mail address: [email protected] (M.M. Walker). 0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2011.08.001

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Microsporidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Giardiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duodenal ulcer and erosions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gastric metaplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eosinophils and mast cells in the duodenum – functional bowel disorders, eosinophilic gastroenteritis and non coeliac food-induced enteropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction Patients presenting for upper gastrointestinal (UGIT) endoscopy to investigate unexplained diarrhea and iron deficiency anemia will usually have duodenal biopsies taken to exclude a diagnosis of coeliac disease. Some particularly in paediatric practice advocate routinely taking biopsies at upper endoscopy to exclude this and other common duodenal diseases although this is controversial [1,2]. While histology remains the gold standard for the diagnosis of coeliac disease, recent developments in serological testing have made this an easier diagnosis, and biopsy may be restricted to confirming the diagnosis [3]. Notably, the diagnosis of established coeliac disease on biopsy is straightforward, but more subtle, and early lesions may pose a challenge. The Marsh classification [4] was an important step forward in permitting histopathologists to diagnose coeliac disease at an early stage prior to substantial damage to the small bowel mucosa. This classification has subsequently been modified by Oberhuber et al. [5] and recently simplified by Corazza and Villanacci [6], to take into account endoscopy of the UGIT with forceps biopsies which allow ready access to the duodenum. Newer endoscopic procedures such as push–pull enteroscopy (balloon enteroscopy) with biopsy allow access to the small bowel beyond the second part of the duodenum, and we need to tailor our diagnosis accordingly [7,8]. Considerable controversy still remains as to what constitutes the normal histology of the duodenum and small bowel. With increased numbers of duodenal biopsies, a new entity, lymphocytic duodenosis (increased intraepithelial lymphocytes with normal villous architecture) has been revealed in patients with negative coeliac serology; this finding is recognized to also be associated with Helicobacter pylori, drugs, autoimmune and other diseases including food allergy [9]. Full thickness small intestinal biopsies can be acquired at laparoscopy for investigation of enteric neuropathies in severe dysmotility disorders, and these specimens may allow a diagnosis and, importantly, prognostic information [10]. In the future, endoscopic techniques may permit similar full thickness specimens to be obtained without the need for surgery [11]. Acid is neutralized in the first part of the duodenum by bicarbonate secreted by Brunner’s glands, and the process of absorption commences. Biopsies are also taken to investigate malabsorption due to suspected infection and metabolic disorders [12]. The World Health Organisation (WHO) has recently placed Giardiasis on the list of neglected diseases; these trophozoites may be seen in the duodenum and can be associated with pathology [13]. Despite highly active anti-retroviral therapy (HAART) immunosuppressed patients are still affected by duodenal pathogens if T lymphocyte counts are low and biopsy plays an important role in diagnosis [14]. The duodenum receives chyme from the stomach – 64 food admixed with acid and pepsin, which has been “churned” by 65 the unique circular musculature of the antrum. Acid is neutralized 66 in the first part of the duodenum by bicarbonate secreted by 67 Brunner’s glands, and the process of absorption commences. 68 Biopsies are also taken to investigate malabsorption due to suspected 69 infection and metabolic disorders [12]. The histology

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of duodenal mucosa in acid-related disorders reflects the damage seen at endoscopy. This is not the case in gastric mucosa, where this can appear normal in the face of severe active and chronic inflammation in, for example, H. pylor-associated gastritis; biopsy here is necessary to assess damage [15]. However, rates of infection of H. pylori are dropping in certain parts of the world with the disappearance of duodenal ulcer disease due to these bacteria [16]. Although the prevalence of duodenal ulcer disease is decreasing, it is still linked to drugs, which may cause ulceration, particularly non steroidal anti-inflammatory drugs (NSAIDs), estimated to cause ulceration in 20% of long term users, equally distributed between the gastric and duodenal mucosa [17]. Recent observations in functional bowel disorders suggest that the duodenum may be a key site for pathology. In functional dyspepsia (FD), patients with early satiety may have excess eosinophil infiltration [18], and the mast cell is probably a key player in the irritable syndrome (IBS) in the duodenum, jejunum and ileum [19].

Coeliac disease Coeliac disease is common compared to original estimates [20] and is believed to be present in up to 1 in 100 of the population although only about 10–15% affected are clinically diagnosed [3]. This is the only autoimmune disease where both the antigen (gluten) and the responsible genes (which are necessary but not sufficient for disease) are known. Dietary immunogenic gluten proteins in wheat, barley and rye reach the lamina propria due to increased intestinal permeability and are deamidated by the enzyme transglutaminase 2. These immunogenic peptides are presented as a complex with HLA-DQ2 or HLA-DQ8 molecules of antigen presenting cells to trigger a T cell response in susceptible individuals, which leads to mucosal damage and subsequent malabsorption [3].

Diagnosis of coeliac disease Biopsies – Where from and how many? Coeliac disease classically manifests in the proximal small intestine and extends variably to the jejunum [21]. In established damage, the endoscopy appearances are typical with flat mucosa which can be targeted for biopsy [22]. However, in early disease, the mucosa may look normal, and it is recommended that biopsies are taken across a circular fold to avoid crushing artifact [22]. While 4–6 biopsies are recommended for diagnosis [23,24] in practice (at most) 4 are usually obtained; expert opinion suggests two from the first part of the duodenum and two from the second part be taken, the bulb being a useful site which mirrors more distal changes [21,25]. Biopsy forceps size makes little difference to diagnostic yield [26], but for histopathologists, larger biopsies are more fruitful as orientation is easier.

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Histological classification

Serology

Architecture, villus crypt ratio and crypt hyperplasia

According to recent NICE guidelines [3] if coeliac disease is suspected clinically, then the first line diagnostic test should be serology rather than biopsy. IgA tissue transglutaminase (tTG) and IgA endomysial antibodies (EMA) serological tests show high levels of sensitivity and specificity in the diagnosis of coeliac disease. Laboratories should use IgA tTG as the first choice test and if this result is equivocal proceed to an IgA EMA test. Similar guidelines are recommended in the USA [40]. In a recent study, it was shown that while tTG is the appropriate first serological test and if used in isolation will detect most cases of coeliac disease (sensitivity 90.9%), this test has a positive predictive value of only 28.6% (because predictive value depends on the disease prevalence) [41]. Therefore, most clinicians would adopt a cost effective two step approach, but need to be aware that this still misses some cases of coeliac cases. In this study, 1/13 cases of coeliac disease were undetected, and a recommendation is made that biopsy should be undertaken in any case where the clinician is suspicious of coeliac disease, despite negative serology. Notably a normal tTG in coeliac patients on a gluten-free diet did not predict recovery of villous atrophy at one year, and it has been recommended that these patients should undergo re-biopsy if symptomatic [41]. IgA deficiency is common in the population (1:131 of patients tested for coeliac disease and 1:6 of those properly evaluated) [42], and investigation for IgA deficiency should be done when the laboratory detects a low optical density (OD) on IgA tTGA test, very low IgA tTGA results or low background on an IgA EMA test [3].

To assess a biopsy appropriately, 3–4 villi with correct orientation are necessary [27]. A cardinal histological feature of coeliac disease is villous atrophy [12,24,27,28]. Normal villi are slender, and the normal crypt: villus ratio is variably cited at 3:1–5:1 [29] but even 2:1 [30], 1.82:1 [31] and 1:1 [32] have been assigned as normal. Villous blunting in coeliac disease is preceded by crypt hyperplasia, lengthening of the crypt depth, as enterocytes have an increased turnover alongside an increase in mitotic figures high in the mucosal surface [12]. Crypt hyperplasia is the initial architectural change seen in dose-related gluten challenge, and is instigated by intraepithelial T lymphocytes [33]. Intraepithelial lymphocytes The intraepithelial lymphocyte (IELs) count/100 enterocytes along villi in coeliac disease is of interest but also subject to controversy as to what constitutes a normal count, and whether the distribution of IELs (tip, sides) is important. How to count? It is most important to have a practical method to count IELs in routine pathology practice. The number of IELs per 100 enterocytes can be counted and the distribution noted. While an even or diffuse distribution of IELs along both the sides and tips of villi are suggestive of coeliac disease in architecturally normal villi, increased IELs at the villus tip are also seen, but are not as common as an even distribution in coeliac disease [34]. The decrescendo sign with increased IELs in the basal part of the villus with loss of IELs along the upper part of the villus and tip is a pointer to normal [35]. Simply counting IELs/20 enterocytes at the tips of 5 villi is a recommended speedy method to assess an increase in IELS and is sensitive and specific [35,36]. How many is too many? The cited normal number of IELs has been quoted as traditionally <40 [37], but as these were calculated in biopsies taken distally in the small bowel by Crosby capsule where IELs are greater in number. It is now acceptable to take <25 [31], and some suggest even 20 [38] as normal in duodenal biopsies seen in routine practice. IELs are mainly cytotoxic ␥␦ T cells, responsible for mucosal damage seen in coeliac mucosa [39]. Simplifying duodenal biopsy reporting of coeliac disease The architectural villus changes are variable and subject to ethnicity, site, biopsy artifact and intraobserver variation, making reporting potentially complex. A sensible proposal by Corazza and Villanacci [6] simplifies the classification of coeliac disease by Marsh and Oberhuber, incorporating these into three main categories: non atrophic (grade A), and atrophic (grade B), grade B to be subdivided into B1 – the villus: crypt ratio less than 3:1 with detectable villi, and B2 with flat mucosa, i.e. partial and total villous atrophy. Non atrophic (grade A) lesions are characterized by an increase in intraepithelial lymphocytes with normal villous architecture. This system is commendable in that as stated in their paper “the greater the number of diagnostic categories of a method the lower the interobserver and intraobserver agreement, with a consequent reduction in its diagnostic reproducibility”. A consensus is needed to adopt these proposals as recommended by Dickson et al. [27]. The problem emphasizes the importance of clinicopathological correlation to diagnose coeliac disease with serology and sometimes the need to proceed to HLA typing (to rule out disease).

HLA typing Despite obtaining a biopsy and serology, there are still individuals with a strong suspicion of having coeliac disease in whom these results may be equivocal. It is then necessary to test for HLA haplotypes which can stratify these patients into high or low risk categories [39,42]. Over 97% of those with coeliac disease will have the DQ2 and/or DQ8 markers, in comparison to 30–40% of the population as a whole [43]. If then a patient is negative for these markers, it is exceedingly unlikely they have coeliac disease [20]. Lymphocytic duodenosis Lymphocytic duodenosis (LD) is defined by normal villous architecture with increased intraepithelial lymphocytes (>20–25/100 enterocytes) and is gaining recognition as a common problem in surgical pathology reporting [9,45]. While the most likely diagnosis is non atrophic gluten sensitivity, there are many causes of increased intraepithelial lymphocytes which are not due to coeliac disease (Table 1). In a retrospective survey in St. Marys Hospital, London, 9% of the duodenal biopsies attained during endoscopy at St. Mary’s NHS Trust in one year were diagnosed as LD, and 23% of these patients subsequently tested positive by serology for coeliac disease. In a large study of 124 patients with LD compared to 454 patients with coeliac disease and villous atrophy, 50% did not belong in the spectrum of coeliac disease as identified by HLA typing and serology. Patients with LD were less likely to have anemia or skin disorders but diarrhea and weight loss were equally distributed [46]. Malabsorption A useful scheme to classify malabsorption is described by Owens [12] in which three broad categories are proposed:

M.M. Walker, N.J. Talley / Pathology – Research and Practice 207 (2011) 538–544 Table 1 Causes of increased (>25) intraepithelial lymphocytes (IELs) in duodenal biopsies. Gluten sensitive enteropathy [9,45] Active coeliac disease Latent and silent coeliac disease Hypersensitivity/intolerance to non-gluten proteins [48,49] Cow’s milk Cereals Eggs Peanuts Soy Autoimmune conditions [9,54] Hashimoto thyroiditis Type I diabetes

Graves disease Rheumatoid arthritis Psoriasis Multiple sclerosis Systemic lupus erythematosus Haemolytic anemia Chronic inflammatory conditions [56,57]

Crohn’s disease Microscopic colitis Collagenous colitis

Drugs [47] Non-steroidal anti-inflammatory drugs (NSAIDs) Proton pump inhibitors (PPIs) Infection [14,50–53] Giardia lamblia Cryptosporidium Viral Tropical sprue H. pylori gastritis Bacterial overgrowth Other immune disorders [55] Glomerulonephritis Hypogammaglobulinaemia due to IgA deficiency or common variable immunodeficiency Graft vs. host disease

Neoplastic disorders [58,59] Enteropathy-associated T-cell lymphoma (EATL) Refractory sprue

1. Maldigestion, e.g. due to gastric insufficiency following gastrectomy and consequent small intestinal bacteria overgrowth (SIBO) or insufficiency of digestive mediators, e.g. bile salt and enzyme deficiencies. 2. Small intestinal mucosal insufficiency such as enteropathy and mural neuromuscular problems 3. Microbial causes, infection and bacterial overgrowth. Small intestinal bacterial overgrowth (SIBO) The overgrowth of bacteria in the small bowel is typified by symptoms of bloating, diarrhea and sometimes malabsorption. It has been defined as >105 colony forming units (cfu)/ml of bacteria in aspirates obtained from the small intestine [60]. Hydrogen breath tests, using glucose or lactulose, are the most commonly used tests for SIBO [61]. The mucosal histology may be normal [62], but may show mild villous atrophy and an increase in IELs [63]. While SIBO has been implicated in irritable bowel syndrome [64], a recent review suggests the role of breath testing for SIBO in individuals with suspected IBS remains unclear [65]. Disaccharidase deficiency The disaccharidases in the small bowel are responsible for breakdown of the sugars lactose, sucrose and maltose. Disaccharidase deficiencies of lactase, sucrase and maltase are described [66]. Lactase deficiency is most common [67]. Lactase is downregulated at weaning, and in some, this decreased enzyme synthesis (to the point of absence) leads to lactose intolerance [68,69]. Intestinal bacteria ferment undigested lactose, and abdominal bloating and discomfort ensues. Intact lactose also causes osmotic diarrhea and intestinal dysmotility [66]. While this may be a primary event in some, secondary damage to the mucosa (commonly post infective) also leads to lactase deficiency. Histology may differentiate primary from secondary disaccharidase intolerances. Low disaccharidase levels with normal histology

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suggest a primary disaccharidase deficiency. Histology can also point to a preceding event such as infection or coeliac disease. Disaccharidase activities vary along the small bowel, with low values in the proximal duodenum, peaking in the mid jejunum, and decreasing activities in the ileum [66]. Histology can vary in primary deficiency, and mean values of lactase, sucrase and maltase values were not significantly different in patients with mild villous atrophy and increased chronic inflammation and focal acute inflammation compared with those with moderate lesions, but were significantly lower in severe lesions compared with moderate lesions [70]. Distribution of lactase on the surface of the villi is intermittent in lactase deficiency, and this can be demonstrated by immunohistochemistry [71]. Hydrogen breath tests can also be used to determine lactose intolerance [61]. An exclusion diet is probably the most cost effective investigation in the first instance. Mural neuromuscular disorders Enteric neuromuscular disease is characterized by aganglionosis, neural degeneration, hyperplasia and dysplasia, inflammatory neuropathy, mitochondrial dysfunction and interstitial cell dysfunction [10] which is usually well beyond the reach of mucosal biopsies. These diseases are usually diagnosed clinically with confirmation by full thickness small intestinal biopsies. All layers of the wall and mucosa are investigated by morphometry, immunocytochemistry and molecular techniques [10,72]. However, a recently described entity, intestinal lymphocytic epithelioganglionitis, deserves mention here as this presents with a lymphocytic enteropathy in the mucosa, which on full thickness biopsy shows myenteric ganglioneuritis accompanied by hyperplasia and hypertrophy of the Cajal cell of in the small intestinal wall [73]. Opportunistic infections and HIV enteropathy Dyspepsia is a major symptom in those infected by HIV taking highly active antiretroviral therapy (HAART) [14]. Pre the HAART era, opportunistic infection (OIs) of the upper GI tract was common and included exotic pathogens [74,75]. Enteropathy with villous atrophy without crypt hyperplasia (indicative of inability to repair the absorptive surface capacity) but with normal immune and goblet cell counts has been described in patients with HIV-AIDS [76]. Promising results with HAART show effective treatment of intractable diarrhea, probably due to restoration of the intestinal defense mechanisms and elimination of pathogens [77]. The stage of immunodeficiency still determines the risk of infection, and the CD4 count and viral load can help predict this [78]. While the proportion of OIs has decreased, these infections (Cytomegalovirus, Cryptosporidium sp., Schistosoma mansoni sp. and Strongyloides stercoralis) occur in a small proportion of HIV patients on HAART with dyspepsia. To diagnose OIs and distinguish infection from functional disorders, it is recommended that multiple biopsies are taken, even from normal-appearing mucosa, at both gastric and duodenal sites [14]. Microsporidia Microsporidia are obligate intracellular protozoa [79] most usually associated with HIV/AIDS infection [80], but have recently been described with increasing frequency in transplant recipients with intractable diarrhea [81] and may also infect immunocompetent travelers [82]. Most often, HIV-infected patients have a very low CD4 count if infected by microsporidiosis, and the prevalence is quite low (1.5%) in these patients [80]. Enterocytozoon bieneusi and the Encephalitozoon spp. currently are the most prevalent microsporidia identified in humans [79]. Usually, the diagnosis is

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made by detection in feces by microscopy and PCR [83], but biopsies particularly from the duodenum may be successful in making a diagnosis. Giardiasis Although G. lamblia is not life-threatening, it is still considered the most common water-borne diarrhea-causing disease [84], and human infection may range from asymptomatic shedding of Giardia cysts to symptomatic giardiasis [85]. Globally, there are 280 million cases per year, and Giardiasis is on the WHO “list of neglected diseases” [13]. Giardiasis may be a zoonosis, but human–human spread is most common. Common symptoms are abdominal pain, nausea and severe watery diarrhea [86]. The histopathology varies in the small intestine, ranging from partial villous atrophy to no visible pathology; only 3.7% of adult cases showed pathology, with mild villous atrophy, crypt hyperplasia and mild inflammation of the lamina propria. Trophozoites are most commonly seen in duodenal mucosa (82.5%), but rarely in gastric antral mucosa (8.7%) jejunal mucosa (2.1%) ileal mucosa (12.1%) and colon (0.4%) [87]. The changes described at a molecular level are a reduction in intestinal disaccharidase activity and intestinal protease activity. Disruption of the microvillous brush border and increased epithelial permeability with bacterial overgrowth are not readily apparent on histology [88]. CD117 (C-kit) has proved to be a useful marker of trophozoites if their presence is suspected [89]. Duodenal ulcer and erosions Known common causes of duodenal ulcer are H. pylori infection [90] and non steroidal anti-inflammatory drugs (NSAIDs) [91]. The prevalence of H. pylori infection is falling in Western nations; overall H. pylori prevalence among many studies has varied from a low of 11% in Sweden to 36% in Iceland [16]. While the consumption of NSAIDs is increasing, it seems prescription of proton pump inhibitors have had a significant effect in decreasing complications from peptic ulcer disease – this may be multifactorial and coincide with decreased smoking habits and the fall in H. pylori infection in the population [92]. In about 2% of patients with duodenal ulcer, despite H. pylori eradication, duodenal ulcers may recur. This group have a significantly higher stimulated acid output than H. pylori-negative healthy controls; it is probable that these patients have an excessive and functioning parietal cell mass in the gastric body, and may need continuing acid suppression even in the absence of infection [93]. While erosions and ulcers may be acid or drug-related, inflammatory bowel disease (IBD), both Crohn’s disease [94] and possibly ulcerative colitis [95] may manifest in the small bowel. Ischaemia also presents as erosions, which may be associated with sickle cell anemia [96]. Other causes of duodenal ulceration include non H. pylori Helicobacters, unrecognized NSAID use, Zollinger Ellison syndrome, cirrhosis and lymphoma [97]. The risk of a false negative in detection of H. pylori is increased with recent proton pump inhibitor use, and although withdrawal is recommended prior to testing, patient compliance may be questionable, and this may also cause problems in identification of bacteria [98]. While biopsy of duodenal ulcers with a clinically obvious cause may be questionable, persistent or recurrent ulceration should be biopsied to exclude other disease [99,100]. Gastric metaplasia Gastric metaplasia (GM) is common in patients infected with H. pylori, particularly those with duodenal ulceration, where rates are

around 55% and in those with distal gastric ulcers, 24%, where GM is uncommon in those with a highly selective vagotomy at 8% [101]. The high acid load to the duodenum probably leads to GM, and this epithelium can be colonised by bacteria [102]. However, GM is not specific to acid injury and is also associated with Crohn’s and coeliac disease [103], but is not associated with cigarette smoking or use of non steroidal anti-inflammatory drugs [104,105]. Eosinophils and mast cells in the duodenum – functional bowel disorders, eosinophilic gastroenteritis and non coeliac food-induced enteropathy Recent studies have suggested that duodenal biopsies may give an insight into the pathogenesis of certain types of functional bowel disorders, namely functional dyspepsia (FD) and irritable bowel syndrome (IBS), and that the duodenum possibly plays a role in contributing to symptoms in these disorders. The traditional view is that functional bowel disorders by definition are not organic diseases, and biopsy is not helpful except to exclude other conditions. Treatment of these conditions remains a considerable challenge [106,107]; only a small benefit is gained by eradication of H. pylori, while use of acid suppression only benefits one third of patients [105]. In both adults and children, duodenal eosinophilia has been shown to be associated with functional dyspepsia (defined as >10/1 HPF for children [107] and >22/5 HPFs with or without clusters of eosinophils at the base of glands for adults [18]). Normal duodenal counts were defined by <10/HPF in children and 19/5 HPF in adults in these studies based on control values [18,107]. The observation of increased eosinophils in functional dyspepsia has been confirmed by studies from Brazil [108] and the US [109]. In IBS, mast cells are significantly increased in the duodenum with a mean of 132 per 5 HPFs in controls and 203 per 5 HPFs in IBS subjects in the first part of the duodenum and 152 per 5 HPFs versus 255 per 5 HPFs in the second part, with no significant difference in eosinophils in this condition [19]. Previous studies show mast cell hyperplasia in the jejunum [110] and ileum [111], and indicate that mucosal inflammation is not limited to the lower small intestine and colon in IBS. It is therefore plausible that eosinophil–mast cell interactions may induce abdominal pain or symptoms related to meals; the inflammatory component may be triggered by pathogens or allergens in contact with the small bowel mucosa. These components of the innate immune system may then interact with the enteric and central nervous system to cause symptoms [112]. Gastroduodenal eosinophilia is also seen in the primary eosinophil disorder eosinophilic gastroenteritis, a rare disease associated with allergy [113]. True food allergies occur in about 1–2% of the population – mainly children – and common provoking allergens include peanuts, soy, tree nuts, cow’s milk and egg [114,115]. The differential diagnosis of duodenal eosinophilia also includes helminth infection [116] and inflammatory bowel disease [117]. Duodenal eosinophilia has also been observed in patients with allergic airways disease undergoing elective endoscopy [118]. Summary and conclusions When duodenal biopsies are taken in patients with diarrhea and the clinicians’ question is whether this patient has coeliac disease, it is prescient of the pathologist to think of the differential diagnosis. Serology for coeliac disease is a cost effective test prior to biopsy, and this should be suggested if this has not been done. Other causes of “coeliac histology” include disorders of malabsorption. Pathogens may be evident. Drug related pathology may be apparent, particularly over the counter medication which patients may not declare. Even functional bowel disorders may prove to have a

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specific duodenal pathology appertaining to the eosinophil–mast cell axis, with eosinophilia in functional dyspepsia and mast cells in IBS.

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