From 1997 to 2007, a prospective study was conducted on 85 patients with ulcerative colitis who were admitted to the Medicine department of V. Cervello Hospital in Palermo due to a severe colitis attack (according to the Truelove-Witts criteria) (Figure ). All patients were treated with conventional and standardized corticosteroid treatment (1 mg/kg per day) and were endoscopically evaluated with a different approach: from 1997 to 1999 sigmoidoscopy was performed only in steroid resistant patients (17 patients) whereas from 2000 to 2007 sigmoidoscopy was performed in all patients (68 patients) at ward admission, taking systematically rectal biopsies. Therefore a total of 85 patients (Table ) was investigated for HCMV infection with 3 different techniques.
Series studied. HCMV: Human cytomegalovirus; ICH: Immunohistochemical; PCR: Polymerase chain reaction; HE: Hematoxylin and eosin.
Clinical characteristics of the series n (%)
The rectal biopsies were immediately fixed with 10% buffered formalin for 2 h to obtain tissue fixation; afterwards the preparation was subjected to several processes (lavage, dehydration, clearing, paraffin impregnation and embedding) to prepare sections with a thickness of 3-4 μm.
The histologic specimens were examined using the following techniques: (1) Light microscopy with hematoxylin and eosin (HE) stain in order to document the microscopic disease activity and allow the detection of cytomegalic cells, markers of infected viral cells. Cytomegalic cells, which are 2-or 4-fold larger (25-35 μm) than surrounding cells, contain a basophilic intranuclear inclusion (8-10 μm) eccentrically placed and sometimes surrounded by a clear halo giving it an “owl’s eye” appearance, and thickened nuclear membrane, frequently associated with smaller granular intracytoplasmatic inclusions. Intranuclear inclusions were observed in epithelial, endothelial, stromal and smooth muscle cells. A biopsy was regarded as positive by light microscopy for HCMV if a single cell showed intranuclear or cytoplasmic inclusions and cytomegalic characteristics (Figure ); (2) Immunohistochemical (ICH) procedure for HCMV performed on a paraffin- embedded section with monoclonal mouse antibodies anti-Human CMV (clone BM204) and conjugated to a peroxidase-labeled amino acid polymer by peroxidase-antiperoxidase (PAP) method in order to detect viral proteins. Nuclear or cytoplasmic antigen was identified by the typical brown reaction product of the PAP method; and (3) Nested polymerase chain reaction (nPCR): (a) DNA extraction: DNA was extracted from 10 mm sections of paraffin wax embedded tissues. Five sections were cut with a standard microtome from every paraffin wax block and transferred into a 1.5 mL microtube. To prevent cross contamination between the samples, the microtome blade was washed with xylene and ethanol after sectioning of each block. DNA extraction was performed using a conventional method. The conventional method consisted of xylene/ethanol dewaxing followed by overnight proteinase k digestion in lysis buffer. The sample was heated at 95°C for 5 min to inactivate the proteinase K. We checked the quality of samples by PCR for the housekeeping gene b-globin (fragment of 268 bp); and (b) Detection of viral DNA: Nested PCR was used to detect the presence of viral DNA in colon tissues. Two pairs of primers annealed to the gB region of HCMV. Primers used for the first-round product and second-round PCRs are as follows (5' to 3'): first-round primer 1, GAGGACAACGAAATCCTGTTGGGCA; first-round primer 2, GTCGACGGTGGAGATACTGCTGAGG; second-round primer 3, ACCACCGCACTGAGGAATGTCAG, and second-round primer 4, TCAATCATGCGTTTGAAGAGGTA, to obtain a CMV fragment of 100 bp.
Original reproduction of epithelial cytomegalic cells (arrows) in rectal biopsy (hematoxylin and eosin stain, 40 ×).
PCR reaction mixture contained 0.5 U Taq polymerase, 1 × PCR Buffer (50 mmol/L KCl and 10 mmol/L Tris-HCl (pH 8.4), 0.2 mmol/L of each dNTPs, 1.5 mmol/L MgCl2, 10 pmol of each primers and 100-200 ng of extracted DNA. The conditions for the first-round PCR were as follows: denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min followed by 5 min final extension at 72°C.
The conditions for second-round PCR were as follows: denaturation at 94°C for 5 min, followed by 25 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min followed by 5 min final extension at 72°C.
The PCR amplification products were run on 2% agarose gel and stained with ethidium bromide and visualized under ultraviolet light.
All patients were also tested for HCMV-pp65 antigenemia in peripheral leukocytes by CMV-CINAkit (Argene®
Biosoft). The samples were considered positive for active HCMV infection using a cut-off value of 5 positive fluorescent nuclei/2 × 105
]; between 1 to 4 positive cells the result was considered questionable and the blood sample was repeated after 48 h to value a possible increase of cell nuclei positivity. If we detected histopathological examination positive we considered the patient HCMV-infected but not surely with active replication; if both tests (histology and pp65 antigenemia) were positive we judged the patients candidates for antiviral treatment due to a probable active replication. However the last decision about antiviral treatment was related to the disease activity without significant improvement of the disease course after treatment for underlying disease.
Sigmoidoscopy was performed in patients reaching clinical remission in order to control endoscopic activity and the presence of HCMV.
The patients were followed clinically as outpatients quarterly to evaluate the remission time, the number of relapses, the immunosuppressive therapies and the need for surgery.
We define as remission a combination of clinical parameters (stool frequency ≤ 3 per day with no bleeding); the term relapse is used to define a flare of symptoms in a patient with established ulcerative colotis (UC) who is in clinical remission, either spontaneously or after medical or surgical treatment.
In the case of clinical relapse, sigmoidoscopy was performed with multiple biopsies and a search for HCMV in rectal biopsies by the 3 methods and peripheral pp65antigenemia was conducted.
Moreover the asymptomatic patients were examined by colonoscopy every year in order to follow up the natural history of HCMV infection regarding histologic persistence, to understand more clearly the possible role of “bystander” or promoter to the disease relapse.
Oral and written informed consent was obtained from each patient before any procedure.
The rate of surgery and clinical relapse rates in the 2 groups of patients (HCMV positive and negative) were compared using χ2 test.