Nizar N. Ramzan

Diagnosis and monitoring of Whipple disease by polymerase chain reaction.

Whipple disease is a systemic bacterial infection with such protean clinical manifestations as diarrhea; abdominal pain; fever; lymphadenopathy; chronic arthralgias; weight loss; and, occasionally, central nervous system involvement that may include dementia, lethargy, and motor and sensory deficits [1-3]. At the time of diagnosis, most patients present with weight loss, diarrhea, and abdominal pain. Recognition of bacillary organisms in a small-bowel biopsy specimen that are positive on periodic acid testing and negative on acid-fast testing leads to the correct diagnosis. However, patients sometimes present with symptoms for which small-bowel biopsy is not usually done. Systemic manifestations, such as pleuritis, lymphadenopathy, cardiac valvular lesions, fever, and wasting may precede gastrointestinal symptoms [1, 2]; symptoms of arthritis may begin years before Whipple disease is diagnosed [2]. Patients with extraintestinal Whipple disease may present with involvement of the central nervous system or the eye [3-6]. Thus, the disease may remain undiagnosed in many patients who never develop gastrointestinal involvement; patients may also receive other diagnoses, such as rheumatoid arthritis or sarcoidosis, in the intervening period. The greatest challenge is to diagnose cases that appear to be clinically consistent with Whipple disease but for which a histologic diagnosis is either negative or equivocal. Because Whipple disease is uncommon, formal prospective evaluations of therapeutic regimens have not been done. Empirical antibiotic therapy with trimethoprim-sulfamethoxazole, cephalosporins, penicillins, or tetracyclines has varied in duration and outcome. In a case series of 29 patients with Whipple disease, the duration of treatment ranged from 5 days to 4 years [2]. Alleviation of symptoms and correction of biochemical and pathologic abnormalities were not related to the duration of antimicrobial therapy. A review of antibiotic therapy in 88 patients with Whipple disease who had long-term follow-up suggested that clinical relapse occurs in as many as 35% of cases [7, 8]; the outcomes of treatment for relapse of disease at the central nervous system were particularly poor [2, 8]. No test for cure is available for Whipple disease, but the recent identification of the 16S ribosomal RNA (rRNA) of the Whipple-associated bacillus, Tropheryma whippelii, has led to the use of 16S rDNA primers for detection of this organism on polymerase chain reaction (PCR) [9, 10]. Polymerase chain reaction assays with high sensitivities have shown T. whippelii in tissues that show no evidence of disease [9]. In this study, a PCR-based test for the detection of T. whippelii was evaluated. Our results suggest that PCR may be useful for the diagnosis of Whipple disease and for monitoring patients who have been treated with antibiotics, thereby allowing the physician to better direct the duration of antibiotic therapy and, perhaps, predict clinical relapse. Methods Patients Thirty patients who received a diagnosis of Whipple disease at the Mayo Clinic between 1952 and 1994 were identified through a computerized search of a diagnosis database. Medical charts of these patients were reviewed. Characteristics of 29 of the patients have been reported elsewhere [2]. Tissue Samples Tissue samples embedded in paraffin blocks were retrieved from the archives of the Mayo Clinic tissue registry; one to seven samples were available from each patient. The anatomic origin of the samples included the small intestine, rectum, pancreas, liver, brain, spleen, and lymph nodes. Post-treatment biopsy specimens were available from 17 of the 30 patients. The medical records of these 17 patients were reviewed by a gastroenterologist who was blinded to the results of PCR and histologic review. All histologic sections were rereviewed in a blinded manner by one pathologist. In small-intestine specimens, the diagnosis of Whipple disease was confirmed on the basis of the following criteria: 1) presence of macrophages in the lamina propria that contained periodic acid-Schiff staining granules, 2) resistance of periodic acid-Schiff-positive granules to diastase, 3) distortion of villous architecture due to the expanded, infiltrated lamina propria, and 4) dilated lymphatic channels (in some instances). Tissue specimens from regions other than the small intestine were also tested for evidence of Whipple disease. Histologic criteria were the presence of foamy histiocytes with bacilli that were positive on periodic acid-Schiff staining and were resistant to diastase and, in tissues from lymph nodes, dilated sinusoids with a Swiss cheese appearance. A histologic section of the small bowel from 1 patient that had been read elsewhere was reexamined in the Mayo Clinics Department of Pathology to confirm the diagnosis; a whole-blood sample (500 micro L) was obtained from this patient after a week of therapy with trimethoprim-sulfamethoxazole. In addition to the 30 patients who had confirmed cases of Whipple disease, 8 patients who had presented with gastrointestinal symptoms and had had biopsy of the small intestine were identified by using a medical records database. Whipple disease had been considered in the differential diagnosis for all 8 patients before biopsy of the small intestine was done. Biopsy specimens were classified as suspicious for or negative for Whipple disease. Medical charts on 7 patients were available for review. Paraffin-embedded biopsy specimens from the small intestine, colon, and brain of 42 patients who had malabsorption of undetermined cause, the irritable bowel syndrome, or viral encephalitis were used as controls. No histologic evidence of Whipple disease was found in these tissues, and all were negative for periodic acid-Schiff-positive macrophages. The control specimens were processed, interspersed with, and tested in a blinded manner in parallel with specimens from the 37 patients described above. Preparation and Analysis of Specimens We extracted two sections of formalin-fixed, paraffin-embedded tissue, each 25 m thick, that had been obtained from each patient. We used a new section of the knife for each cut to avoid cross-contamination during processing. Sections were deparaffinized with xylene and digested overnight at 55 C with 50 L of proteinase K (20 mg/mL) and sodium dodecyl sulfate (10%) in Tris (10 mmol/L) and EDTA (1 mmol/L). The digested tissue was boiled for 15 minutes to inactivate the proteinase K. Deoxyribonucleic acid was extracted by using a chaotropic lysis method with 200 L of the tissue (Isoquick kit, Orca Research, Bothell, Washington). Design of Primers Primers W3FE and W4RB were modified to improve their specificity for T. whippelii [11]. Primers W3AF and W4AR were designed on the basis of 16S rRNA gene sequence alignments of related organisms so that the region of gene that was amplified would be unique to T. whippelii. The expected size of the W3AF and W4AR amplification product was 160 base pairs. Evaluation of Specificity of Primers W3AF and W4AR We determined the specificity of primers W3AF and W4AR by doing PCR on DNA that had been extracted from clinical isolates of many bacteria: Nocardia brasiliensis, N. otitidiscaviarum, N. asteroides, Mycobacterium tuberculosis, M. kansasii, M. chelonae, M. bovis, M. fortuitum, M. avium-intracellulare complex, Actinomyces bovis, A. viscosus, anaerobic Actinomyces species, Corynebacterium species, Propionibacterium species, Rhodococcus equi, unspeciated Rhodococcus, and Streptomyces griseus. Amplification products were found only in R. equi and N. otitidiscaviarum. Sequencing of amplification products of these two organisms showed four variances in nucleotide bases compared with the T. whippelii sequence: C to T at position 1092, G to A at position 1093, C to T at position 1096, and G to A at position 1098. To ensure specific detection of T. whippelii, an 18-mer oligonucleotide probe homologous to T. whippelii sequence was designed to encompass the four-nucleotide variance (see Appendix Table (Table 5)). Table 5. Appendix Table. Sequence of Polymerase Chain Reaction Primers and 16S Ribosomal RNA Positions Relative to Escherichia coli Position 1 Primers W3FE and W2RB, which are also specific for T. whippelii, were used in accordance with the methods described elsewhere [9, 12]. Extraction Control We used the -globin gene, which is found in all nucleated cells, to assess the efficiency of the extraction of DNA from tissue. Tissue samples that were negative for Whipple-associated bacillus 16S rDNA products were amplified for a segment of the human -globin gene. Samples that did not have a positive result when tested for -globin were considered inadequate. Primers GH20 and PCO4 were used for -globin amplification [13]. Polymerase Chain Reaction with Primers W3AF and W4AR An amplification reaction mix, 45 L, was prepared from 10 L of 50% glycerol (10% final concentration), 5 L of 10 x Perkin-Elmer buffer (containing 100 mmol of Tris-HCl per L [pH, 8.3] [Sigma, St. Louis, Missouri], 500 mmol of KCl per L, and 15 mmol of MgCl2 per L) (Applied Biosystems, Perkin-Elmer, Foster City, California), 2 L each of the four deoxyribonucleoside triphosphates, 0.25 units of AmpliTaq polymerase (Applied Biosystems, Perkin-Elmer), 5 pmol of primers W3AF and W4AR, 0.33 L of isopsoralen (25 g/mL), and 19.42 L of sterile water. We added 10% or 5 L of extracted DNA from the specimen to this master mix for a final reaction volume of 50 L. Thermocycling was done as follows. Initial melting was done at 94 C for 2 minutes. This was followed by 50 cycles of denaturing at 94 C for 30 seconds, annealing at 60 C for 45 seconds, and extension at 72 C for 60 seconds. Final extension was done at 72 C for 4 minutes. After each amplification, the microtubes were exposed to ultraviolet light to promote covalent crosslinking of the isopsoralen [14]. This process prevents PCR product contamination, which can

The diagnostic yield of stool pathogen studies during relapses of inflammatory bowel disease

Goals We sought to determine the yield of stool analysis for bacterial culture, ova and parasites, and Clostridium difficile toxin in suspected relapses of inflammatory bowel disease (IBD). Background The diagnostic yield of such stool studies has not been examined recently in the United States. Study The medical records of consecutive IBD patients who underwent stool testing for relapses at our institution between July 1, 2000, and November 25, 2001, were abstracted for demographics, stool test results, recent antibiotic exposure, and hospitalization. Results Fifty-four patients were evaluated during 62 relapses with 99 stool samples. Twelve stool tests were positive. C. difficile accounted for the majority of positive tests (10/12). Of these, 9 (90%) were associated with antibiotic use in the prior month versus 10 (22%) in the C. difficile-negative group (P < 0.001). Hospitalization, prednisone use, or sulfasalazine use did not differ significantly with C. difficile status. Eight C. difficile-positive patients improved clinically with targeted antibiotic therapy. Two bacterial cultures (4%) were positive for Campylobacter jejuni and Plesiomonas shigelloides. Conclusion Stool studies yielded a pathogen, mainly C. difficile, in 20% of the relapsing IBD patients. Antibiotic use was significantly associated with a positive C. difficile toxin. Toxin-positive patients improved clinically with targeted antibiotics.

Clinical significance of granuloma in Crohn's disease

Crohns disease (CD) is diagnosed from information obtained clinically, pathologically, and radiologically. One important pathologic finding is a granuloma, which is helpful when a positive diagnosis of CD will affect treatment. Whether the presence of a granuloma has any clinical implication is not clear. We conducted a retrospective study to determine whether a granuloma found on a biopsy sample is associated with disease severity, fistulizing or perianal disease, frequent relapses, and extraintestinal manifestations. Eighty-two patients were identified who had a biopsy or bowel resection for CD between 1990 and 1994 at a tertiary referral center; 21 (25.6%) had a granuloma. This group was compared with a group of 61 patients without a granuloma. Forty-five percent were male (n = 37), mean age at diagnosis was 42.6 years (median, 39.5 years), mean disease duration at presentation was 8.8 years (median, 4.8 years), and mean follow-up duration was 2 years (range, 1 day to 10.2 years). No significant differences were demonstrated between the two groups by the Fisher exact test with regard to fistulizing or perianal disease, oral aphthous ulcers, disease severity, axial or peripheral arthralgia, episcleritis, anterior uveitis, erythema nodosum, or pyoderma gangrenosum.

Relapse of Inflammatory Bowel Disease Associated With Use of Nonsteroidal Anti-Inflammatory Drugs

To determine whether an association exists between relapse in inflammatory bowel disease and use of nonsteroidal anti-inflammatory drugs (NSAIDs), a retrospective records review was conducted of patients with Crohns disease, ulcerative colitis, or indeterminate colitis examined at an outpatient tertiary care center between July 17, 2000, and November 1, 2001. Extracted data collected during the patients last visit included medication use, maintenance therapy, disease activity, and smoking status. Use of NSAIDs was defined as a daily dose or more of any type the month before relapse. Of 60 patients (22, relapse; 38, remission), 9 (41%) in relapse and 10 (26%) in remission used NSAIDs. Maintenance therapy varied from 68% (relapse) to 92% (remission). The adjusted odds ratio between medication use and relapse was 6.31 (95% confidence interval, 1.16–34.38; P = .03). Use of NSAIDs was associated with relapse. A prospective cohort study that corrects for maintenance therapy is needed to evaluate this relationship.

Colonic perforation in unsuspected amebic colitis.

Unsuspected amebic colitis presenting as inflammatory bowel disease, as in our patient, has been previously reported (4, 7, 8). Misdiagnosis, delay in antibiotic treatment, and institution of immunosuppression were the result of failure to identify the parasite in stool specimens and have resulted in suffering, morbidity, mortality, and surgery. In all previously reported cases, routine stool studies failed to identify E. histolytica (4, 7, 8). The correct diagnosis was only established after reviewing the surgical specimen or biopsies obtained endoscopically. Because the erroneous diagnosis of inflammatory bowel disease can lead to disastrous complications, it is imperative to exclude amebic colitis prior to undertaking steroid therapy, especially in patients with a prior history of travel to or residence in areas with endemic E. histolytica (17). We recommend obtaining at least three stool specimens for microscopic examination, as well as testing for serum amebic antibody. Patients should submit fresh stool specimens directly to the laboratory to allow for prompt diagnostic evaluation. Such an approach might lead to the improved diagnosis of amebiasis.

TRAVELER'S DIARRHEA

This article presents a review of causes, presentation, and diagnosis of travelers diarrhea. Treatment and prevention of this common problem is described in some detail. Finally, a practical and cost-effective approach to evaluating and treating a returning traveler is presented.

Sporadic duodenal gastrinoma presenting as Zollinger-Ellison syndrome requiring surgical treatment

Sporadic duodenal gastrinoma presenting as Zollinger-Ellison syndrome requiring surgical treatment

Efficacy of mycophenolate mofetil in moderate to severe ulcerative and Crohn's colitis—a pilot study

Efficacy of mycophenolate mofetil in moderate to severe ulcerative and Crohns colitis—a pilot study