Comparing virtual with conventional microscopy for the consensus diagnosis of Barrett's neoplasia in the AspECT Barrett's chemoprevention trial pathology audit.

AIMS: To compare the diagnostic accuracy of conventional versus virtual microscopy for the diagnosis of Barrett's neoplasia. METHODS AND RESULTS: Sixty-one biopsies from 35 ASPirin Esomeprazole ChemopreventionTrial (AspECT) trial patients were given a Barrett's neoplasia score (1-5) by a panel of five pathologists using conventional microscopy. Thirty-three biopsies positive for neoplasia were digitized and rescored blindly by virtual microscopy. Diagnostic reliability was compared between conventional and virtual microscopy using Fleiss' kappa. There was substantial reliability of diagnostic agreement (κ = 0.712) scoring the 61 biopsies and moderate agreement scoring the subgroup of 33 'positive' biopsies with both conventional microscopy (κ = 0.598) and virtual microscopy (κ = 0.436). Inter-observer diagnostic agreement between two pathologists by virtual microscopy was substantial (κ = 0.76). Comparison of panel consensus neoplasia scores between conventional and virtual microscopy was almost perfect (κ = 0.8769). However, with virtual microscopy there was lowering of the consensus neoplasia score in nine biopsies. CONCLUSIONS: Diagnostic agreement with virtual microscopy compares favourably with conventional microscopy in what is recognized to be a challenging area of diagnostic practice. However, this study highlights possible limitations for this method in the primary diagnostic setting.

Multicentre review of lymph node harvest in colorectal cancer: are we understaging colorectal cancer patients?

BACKGROUND: Lymph node examination in colorectal cancer is of vital importance for accurate staging. Patients who have fewer nodes examined may be understaged and not offered adjuvant chemotherapy. The national institute of clinical excellence and the association of coloproctology of Great Britain and Ireland both recommend that 12 nodes should be examined for accurate staging. The aim of this study was to assess lymph node harvest at five hospitals in the northwest of England in respect to these guidelines. MATERIALS AND METHODS: This study is a retrospective review of all colorectal cancer resections over a 1-year period at five hospitals. RESULTS: Two hospitals met the national guidelines of a median of 12 or more nodes. Overall, over 50% of colorectal cancers contained fewer than 12 nodes. Fifty-three point seven percent (53.7%) of Dukes B patients did not have 12 nodes in their specimens and may therefore be understaged. There was a significant variation between hospitals in terms of the number of cancers with 12 or more nodes (P < 0.0001) and the number of Dukes B cancers with 12 or more nodes (P < 0.008). CONCLUSION: Over 50% of all colorectal cancer specimens contain fewer than 12 lymph nodes despite clear national guidelines. This is of particular importance to Dukes B cancers where over 53% of cases may be understaged and not offered adjuvant therapy. Significant variation exists between hospitals within the same region.

Prospective study of cyclin D1 overexpression in Barrett's esophagus: association with increased risk of adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma commonly arises from a precancerous condition, Barrett's esophagus, in which the normal squamous epithelium is replaced by a columnar cell-lined epithelium. Genetic alterations occurring in this process could serve as biomarkers for the risk of malignant progression, improve surveillance, and contribute to early diagnosis. We examined two potential biomarkers, cyclin D1 and p53, in a prospective cohort of Barrett's esophagus patients. METHODS: A total of 307 patients were enrolled in an endoscopic surveillance cohort, and esophageal biopsy specimens were collected at each endoscopy. Incident cases of adenocarcinoma were matched to control patients within the cohort by duration of follow-up, age, sex, and length of columnar cell-lined epithelium at recruitment. Biopsy specimens were analyzed for cyclin D1 and p53 protein levels by immunohistochemistry. Statistical tests were two-sided. RESULTS: A total of 12 cases of adenocarcinoma occurred within the follow-up period, and tumor biopsy specimens from 11 cases stained positive for cyclin D1. Biopsy specimens from eight of these patients taken at recruitment also stained positive for cyclin D1. A case-control analysis of biopsy specimens obtained at recruitment revealed a statistically significantly increased risk of progression to adenocarcinoma in Barrett's esophagus patients whose biopsy specimens were cyclin D1 positive (odds ratio [OR] = 6. 85; 95% confidence interval [CI] = 1.57-29.91; P =.0106) but not in patients whose biopsy specimens were p53 positive (OR = 2.99; 95% CI = 0.57-15.76; P =.197). CONCLUSIONS: Cyclin D1-positive staining could be a useful biomarker in identifying Barrett's esophagus patients at high risk of esophageal adenocarcinoma. Given the complexity of genetic alterations in the natural history of this cancer, additional biomarkers will be required to increase the sensitivity and specificity of molecular diagnosis.

Dermatomyositis and Whipple's disease.

A 14-year-old boy presented with a 3-year history of a skin rash typical of juvenile dermatomyositis, and a 2-month history of mild proximal weakness, myalgia, and weight loss. A quadriceps biopsy showed perifascicular fibre atrophy, focal necrosis and regeneration, immunohistochemical labelling for HLA-1 on the surface of the fibres, and focal C5-9 deposition in capillaries. Macrophages with diastase-resistant, PAS-positive cytoplasm were present. Ultrastructural studies showed electron dense and membranous debris. The patient's symptoms responded to intravenous immunoglobulin and oral prednisolone. Four months after discontinuing prednisolone, the patient developed cardiac failure, ventricular tachycardia, and a recurrence of his rash. The 16S ribosomal RNA specific for Tropheryma whippelii was identified by polymerase chain reaction (PCR) analysis in skeletal and cardiac muscle. The myalgia and skin rash responded to prednisolone and oral co-trimoxazole, and the tachycardia is controlled by oral verapamil. This patient appears to have a novel association of juvenile dermatomyositis and Whipple's disease.

The optimal antibiotic combination in a 5-day Helicobacter pylori eradication regimen.

BACKGROUND: Current guidelines for Helicobacter pylori eradication recommend 7 days of a proton-pump inhibitor, clarithromycin (C), and either metronidazole (M) or amoxycillin (A). A shorter course would be cheaper and could be as effective. AIM: This study was designed to investigate the efficacy of three 5-day regimens based on lansoprazole (L). METHODS: 168 dyspepsia patients with H. pylori infection were randomized to receive a 5-day course of either LCM, LAC or CALM, and a 13C-urea breath test was performed after 4 weeks to assess eradication. RESULTS: 160 patients completed the study. Intention-to-treat eradication rates were as follows: LCM 81%, LAC 59%, CALM 88%. LCM and CALM gave significantly better eradication rates than LAC. There was no significant difference in adverse events across the three groups. Logistical regression analysis showed that the specific regimen used and the age of the patient were the only factors influencing eradication outcome. CONCLUSIONS: Five days of CALM yields acceptable eradication rates, and is cheaper than conventional 7-day proton pump inhibitor-triple therapy. It appears to offer good results in metronidazole-resistant strains of H. pylori. A randomized trial comparing 5-day CALM with conventional 7-day therapy is needed before this regimen can be recommended for routine use.

Whipple's disease revisited.

Whipple's disease has traditionally been considered to be a rare multisystem disorder dominated by malabsorption. The recent identification of the Whipple's disease bacillus has, using polymerase chain reaction based assays, fueled advances in the investigation, diagnosis, and management of this disease. This leader reviews the aetiology, clinical manifestations, investigation, and treatment of Whipple's disease in the light of this new information.

Clinical and histological associations of cagA and vacA genotypes in Helicobacter pylori gastritis.

AIMS: To determine the relation among the cytotoxin associated gene (cagA) and vacuolating cytotoxin gene (vacA) status of Helicobacter pylori isolates, the associated clinical diseases, and the severity and pattern of chronic gastritis. METHODS: Helicobacter pylori was cultured from gastric biopsies obtained from dyspeptic patients. DNA was extracted from the isolates and the cagA and vacA status determined by the polymerase chain reaction (PCR). The prevalence of the different cagA and vacA genotypes in three clinical groups, duodenal ulcer, gastric ulcer, and non-ulcer dyspepsia was compared. The histological features in sections from two antral and two corpus biopsies were graded by one blinded observer. The grades were compared with age and sex matched groups with different cagA and vacA genotypes, and with duodenal ulcers, or non-ulcer dyspepsia. RESULTS: Isolates from 161 patients were included. One hundred and nine (68%) harboured a cagA+ strain and 143 (89%) harboured a vacA s1 strain. The prevalence of cagA+ strains in duodenal ulcer patients (94%) was highly significantly greater than in those with non-ulcer dyspepsia (56%). However, of the patients infected with a cagA+ strain, almost equal numbers had non-ulcer dyspepsia or peptic ulceration. Chronic inflammation, polymorph activity, surface epithelial degeneration, atrophy, and intestinal metaplasia were all significantly more severe in the cagA+ than in the cagA- group, whereas only corpus epithelial degeneration was significantly more severe in the vacA s1 group compared with the vacA s2 group. Patients infected with cagA+ strains were almost four times more likely to have antral intestinal metaplasia than cagA- patients. An antral predominant gastritis was present in duodenal ulcer patients compared with matched non-ulcer dyspepsia patients, but this was not attributable to cagA or vacA status. CONCLUSIONS: Helicobacter pylori strains showing cagA positively and the vacA s1 genotype are associated with more severe gastritis but these virulence factors do not appear to determine the overall pattern. The pattern is closely linked to clinical disease. Therefore, it is likely that the nature of the disease complicating chronic infection is determined by host and environmental factors, while bacterial factors determine the magnitude of the risk of developing such disease.

Identification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR.

AIMS: To determine the prevalence of Helicobacter pylori colonisation in the mouths of patients with H pylori gastritis. METHODS: A nested polymerase chain reaction test for the 16S ribosomal RNA gene of H pylori was used on saliva, dental plaque, gastric juice and gastric biopsy specimens from patients attending a dyspepsia clinic. RESULTS: Thirteen patients had histologically confirmed Helicobacter associated gastritis. Twelve of these had positive gastric aspirates by PCR. Five had at least one positive oral specimen. Eight patients with normal gastric biopsy specimens had no PCR positive oral specimens or gastric aspirates. All, however, had PCR positive gastric biopsy specimens. In an attempt to determine the origin of these positive results in normal patients, it was shown that biopsy forceps could contaminate specimens with DNA from previous patients. CONCLUSION: The demonstration of the organism in the mouths of a substantial proportion of dyspeptic patients has major implications for the spread of H pylori and identifies a potential source for reinfection following eradication of the organism from the stomach.

Correlation between epithelial cell proliferation and histological grading in gastric mucosa.

AIM: To determine if there is a correlation between the histological findings in the gastric mucosa and the degree of cell proliferation in gastric antral biopsies. METHODS: Cell proliferation in gastric antral biopsies was determined by in vitro bromodeoxyuridine labelling. Histological sections were assessed using the Sydney System. RESULTS: There was a positive correlation between antral mucosal cell proliferation and the acute inflammatory cell infiltrate (r = 0.29; p = 0.03). There was a stronger correlation with the chronic inflammatory cell infiltrate (r = 0.53; p < 0.0001) and the density of H pylori colonisation (r = 0.54; p < 0.0001). There was no correlation between gastric epithelial proliferation and the degree of atrophy. Stepwise multiple regression indicates that the only independent predictor of epithelial cell proliferation is the density of H pylori colonisation (p < 0.0001). CONCLUSIONS: H pylori increases gastric epithelial cell proliferation through the mucosal inflammatory response and probably by other means. The strong correlation between epithelial proliferation, the chronic inflammatory cell infiltrate, and the density of H pylori colonization may have implications for gastric carcinogenesis.

Whipple's disease without malabsorption: new atypical features.

The diagnosis of Whipple's disease in the absence of intestinal involvement is difficult and often overlooked. We describe five patients aged 8-71 years with normal jejunal biopsies and disparate clinical features, previously unrecognized in Whipple's; all were investigated at a single institution over a period of 18 months. Routine histological examination for periodic acid-Schiff (PAS) positive macrophages and polymerase chain reaction (PCR) analysis for Tropheryma whippelii was performed on the small intestine in all patients. PCR analysis was also performed on various tissues including peripheral blood, lymph node, muscle, synovium and spleen in individual patients. Patients 1, 2, 4 and 5 had unusual presenting features not previously associated with Whipple's: intractable immune thrombocytopenic purpura (ITP), juvenile chronic arthritis, isolated muscle weakness and quadriparesis, respectively. Patient 3 presented with pyrexia of unknown origin. All patients had histologically normal small-bowel biopsies with no evidence of PAS positive macrophages. PCR for T. whippelii was positive in all patients in one or more tissues: peripheral blood, intestine, muscle, lymph node and synovium. PAS-positive macrophages were found in 4/5 patients in various sites: lymph node, muscle, spinal cord. Whipple's disease presents with protean clinical features and should be considered in granulomatous disorders of unknown aetiology even in the absence of gastrointestinal involvement.

Relevance of antibiotic sensitivities in predicting failure of omeprazole, clarithromycin, and tinidazole to eradicate Helicobacter pylori.

Omeprazole 20mg once (od) or twice daily (bd), clarithromycin 250mg bd, and tinidazole 500 mg bd for 7 days (OCT) is an effective regimen against Helicobacter pylori, but the effect of 5-nitroimidazole resistance is unclear. We aimed to evaluate this using the disc diffusion technique and E-test to assess 5-nitroimidazole resistance. H. pylori was cultured from antral biopsies of infected patients as determined by 13C-urea breath test (13C-UBT), histology, and/or rapid urease test. Patients were prescribed OCT, and H. pylori eradication was assessed by 13C-UBT at least 4 weeks after completion of therapy. Antibiotic sensitivities to metronidazole and clarithromycin were evaluated by the disc diffusion method and by minimum inhibitory concentration (MIC) using the E-test. One hundred and forty-one H. pylori-infected patients were enrolled into the study and the organism was successfully cultured in 119 patients (84%). The overall eradication rate was 125/141 (89%). OCT was successful in 62/69 (90%) patients harboring fully sensitive strains of H. pylori compared with 42/45 (93%) of patients with strains that were resistant to metronidazole alone (P = 0.74, Fisher's exact test). MIC was assessed in 22 samples. Using a cut-off point of > 32 microg/ml to define metronidazole resistance, eradication rates were higher against sensitive (9/12; 75%) compared with resistant (3/10; 30%) strains (P = 0.08, Fisher's exact test). 5-Nitroimidazole resistance assessed by the disc diffusion technique is not helpful in predicting OCT failure, but the E-test may be of value.

Relevance of antibiotic sensitivities in predicting failure of omeprazole, clarithromycin, and tinidazole to eradicate Helicobacter pylori.

Omeprazole 20 mg once (o.d.) or twice daily (b.d.), clarithromycin 250 mg b.d., and tinidazole 500 mg b.d. for 7 days (OCT) is an effective regimen against Helicobacter pylori, but the effect of 5-nitroimidazole resistance is unclear. We aimed to evaluate this using the disc diffusion technique (Mast Diagnostics, Bootle, UK) and E-test (Cambridge Diagnostics Services, Cambridge, UK) to assess 5-nitroimidazole resistance. H. pylori was cultured from antral biopsies of infected patients, as determined by 13C-urea breath test (13C-UBT), histology, and/or rapid urease test. Patients were prescribed OCT and H. pylori eradication was assessed by 13C-UBT at least 4 weeks after completion of therapy. Antibiotic sensitivities to metronidazole and clarithromycin were evaluated by the disc diffusion method and by the measurement of minimum inhibitory concentration (MIC) using the E-test. One hundred and forty-one H. pylori-infected patients were enrolled in the study and the organism was successfully cultured from 119 patients (84%). The overall eradication rate was 125/141 (89%). OCT was successful in 62/69 (90%) patients harboring fully sensitive strains of H. pylori, compared with 42/45 (93%) of patients with strains that were resistant to metronidazole alone (P = 0.74, Fisher's exact test). MIC was assessed in 22 samples. Using a cut-off point of >32 microg/ml to define metronidazole resistance eradication rates were higher against sensitive (9/12; 75%) than resistant (3/10; 30%) strains (P = 0.08, Fisher's exact test). 5-Nitroimidazole resistance assessed by the disc diffusion technique is not helpful in predicting OCT failure, but the E-test may be of value.

Do patients need to fast for a 13C-urea breath test?

OBJECTIVE: The 13C-urea breath test (13C-UBT) is a useful non-invasive method of diagnosing Helicobacter pylori infection. One of its limitations, however, is that patients have to fast for 4 h before testing. We have compared the accuracy of a non-fasting 13C-UBT (NF13C-UBT) with a fasting 13C-UBT (F13C-UBT) test and against a gold standard. DESIGN: An unblinded prospective crossover study. METHODS: H. pylori status was assessed by histology, culture and rapid urease test. Patients were defined as H. pylori positive if two or more tests gave a positive result and negative if all tests were negative. H. pylori status was indeterminate if only one test gave a positive result. Following endoscopy patients had a F13C-UBT and then a further NF13C-UBT up to 14 days later after eating two slices of toast with jam or honey and tea or coffee. RESULTS: Of the 222 patients recruited to the study, 123 were gold standard H. pylori positive and 94 were negative with five patients having indeterminate status. Compared to this gold standard the NF13C-UBT had a 98% sensitivity and 96% specificity and the F13C-UBT had a 96% sensitivity and 97% specificity. The NF13C-UBT and F13C-UBT agreed in 217/222 (98%) cases. CONCLUSION: Relaxation of the fasting state does not reduce the accuracy of the 13C-UBT, making this test more convenient for patients.

The Detection of H. pylori by the Polymerase Chain Reaction.

A number of methods are currently available for the detection of Helicobacter pylori, including serology, culture, histology, and isotope breath tests. All have relative advantages and disadvantages of sensitivity, specificity, convenience, expense, and immediacy. The polymerase chain reaction (PCR) was an obvious choice of test once the technique became available to many laboratories. PCR tests for various bacteria, including H. pylori, are available on a wide range of specimens, including cultures and direct clinical samples. The ideal PCR test for H. pylori would be highly sensitive and specific, cheap if performed in bulk, and could produce results much faster than would be possible with culture.

Testicular seminoma metastasizing to palatine tonsil.

Patients presenting with Stage I seminoma of the testis have an excellent prognosis after treatment by orchidectomy and prophylactic radiotherapy to the paraaortic and pelvic lymph nodes. Only 2% subsequently recur but relapse in these cases has been reported in unusual sites such as the prostate and mesentery. We report a case of Stage I seminoma relapsing in another rare site for secondary malignant deposits, the palatine tonsil. This case further illustrates the favourable prognosis for this tumour even in cases of distant recurrence.

Carcinoma arising within an anal gland.

Anal gland carcinoma (AGC) is rare, and its innocuous presentation and developing immunohistochemical profile make the diagnosis of it challenging. Predominant presenting symptoms include anal pain, rectal bleeding and the presence of a perianal mass in advanced stages of the disease. Histological profile commonly reveals an intramural adenocarcinoma with normal unaffected overlying anorectal mucosa. Immunohistochemical analysis shows positive staining for cytokeratin (CK) 7 and negative staining for CK20. MUC5AC expression with CK5/6 and p53 negativity has been reported. The authors report a case of a 68-year-old woman with a rapidly advancing AGC and review the current literature with respect to diagnosis and current consensus on therapeutic management.

Morphometric analysis of fine needle aspirates from breast lesions.

In an attempt to discover the morphometric variables with the most diagnostic power in the differentiation of benign from malignant breast disease, 20 unequivocally benign and 20 unequivocally malignant and histologically confirmed breast aspirates were examined on an image analyser. It was found that standard deviation of nuclear area was the most discriminant variable. Then 23 aspirates initially diagnosed as 'suspicious of malignancy' were measured by the same technique, and standard deviation of nuclear area correctly differentiated all but three cases.

Adaptation of Helicobacter pylori to aerobic growth.

Two of 23 strains of Helicobacter pylori adapted from microaerobic to aerobic growth on blood agar plates incubated in humidified air. The air-adapted strains remained urease and phenylalanine deaminase positive and did not require the buffering effect of an enriched CO2 atmosphere for growth. The significance of this phenomenon remains to be determined as the two strains capable of aerobic metabolism were laboratory-adapted.

Mycobacterium paratuberculosis DNA not detected in Crohn's disease tissue by fluorescent polymerase chain reaction.

The role of mycobacteria in the aetiology of Crohn's disease has been a contentious subject for many years. Mycobacterium paratuberculosis is known to cause a chronic granulomatous enteritis in animals (Johne's disease) and has been implicated as a possible infectious cause of Crohn's disease. However this fastidious organism is only rarely detected by conventional microbiological techniques. This study used oligonucleotide primers to the species-specific M paratuberculosis IS900 DNA insertion element and the polymerase chain reaction to amplify any M paratuberculosis DNA from intestinal tissue DNA extracts. One oligonucleotide primer was fluorochrome-labelled and the presence of fluorescent amplified product was determined using an automated DNA sequencer with a computerised gel-scanning laser. This method was shown capable of detecting 1-2 mycobacterial genomes. Intestinal tissue samples were obtained from 68 patients with histologically confirmed Crohn's disease, 49 patients with histologically confirmed ulcerative colitis, and 26 non-inflammatory bowel disease controls. In no case was M paratuberculosis detected in any of the inflammatory bowel disease tissue samples and only one non-inflammatory bowel disease case was positive. These results do not support the hypothesis that M paratuberculosis has an aetiological role in Crohn's disease.