Investigation of Tissue Cytomegalovirus (CMV) Status Via Polymerase Chain Reaction and Serological Tests in Patients Undergoing Colonoscopy and Intestinal Biopsy
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 139
- 试验地点
- 1
- 主要终点
- Tissue and serum CMV Viral Load
研究概览
简要总结
Introduction and Objectives Patients diagnosed with Inflammatory Bowel Disease (IBD) carry a significantly elevated risk for opportunistic infections and the reactivation of latent pathogens, most notably Cytomegalovirus (CMV). CMV reactivation in the colonic mucosa can exacerbate underlying IBD, leading to poor clinical outcomes and resistance to standard immunosuppressive therapies.
This prospective study was designed with a multi-faceted objective:
To evaluate and quantify the CMV viral load within the intestinal tissues of a pediatric cohort, comprising both IBD and non-IBD control groups.
To identify specific clinical and biological factors associated with increased CMV detection.
To establish potential diagnostic threshold values for CMV viral load in the colonic mucosa, with a primary focus on Ulcerative Colitis (UC) patients-where CMV colitis is most prevalent-as well as patients with other intestinal pathologies.
To perform a comparative analysis of diagnostic modalities, specifically evaluating the diagnostic value of histopathology, serum Cytomegalovirus-Polymerase Chain Reaction (CMV-PCR), and tissue CMV-PCR.
2. Methodology and Procedural Framework The research was conducted at the Pediatric Gastroenterology Endoscopy Unit between May 2022 and March 2024. The study population consisted of pediatric patients undergoing scheduled colonoscopies.
Biopsy Protocol: During the endoscopic procedure, two biopsy samples were systematically obtained from the rectal mucosa. For patients presenting with mucosal ulcers, samples were extracted directly from the ulcerated site; in patients with macroscopically normal mucosa, samples were taken from standard rectal tissue.
Contamination Prevention: To ensure molecular integrity and prevent cross-contamination, separate forceps were utilized for samples intended for PCR analysis versus those intended for histopathology.
Laboratory Analysis: Concurrently, venous blood samples were collected to analyze CMV serology (anti-CMV Immunoglobulin G [IgG] / Immunoglobulin M [IgM]) and serum CMV-PCR. In cases of IgM positivity, CMV IgG avidity tests were performed to distinguish between primary infection and reactivation.
Diagnostic Criteria: A diagnosis of CMV colitis was established based on clinical symptoms, histopathological evidence (Hematoxylin and Eosin [HE] staining and Immunohistochemistry [IHC]), and a serum CMV-PCR threshold of ≥1000 copies/mL.
3. Molecular and Histopathological Techniques Deoxyribonucleic Acid (DNA) Extraction: Tissue DNA was extracted using the QIAamp DNA Mini Kit, involving overnight incubation at 56°C for complete tissue digestion. Plasma DNA extraction was automated via the QIAsymphony SP platform. Internal controls were used in every run to validate extraction efficiency.
Real-Time Polymerase Chain Reaction (Real-Time PCR): Amplification targeted a 105-bp region of the CMV genome using the Artus CMV QS-RGQ Kit. The assay provided a wide linear range (79.4 to 1×10⁸ copies/mL) with a high analytical sensitivity of 42.5 copies/mL. Results were reported as copies/mL for blood and copies/mg for tissue.
Immunohistochemistry (IHC): Four-micrometer tissue sections were analyzed for CMV expression using the Ventana Benchmark XT platform. Nuclear staining was the primary indicator for CMV positivity, with known positive colon mucosa serving as the control.
4. Statistical Analysis Data were analyzed using IBM SPSS Statistics Version 20.0. The normality of the data was verified using Shapiro-Wilk tests and visualization tools (histograms and Q-Q plots).
Comparative Statistics: Categorical variables were assessed via Chi-square tests. Continuous variables were analyzed using Student's t-test (parametric) or the Mann-Whitney U test (non-parametric).
Correlations: Relationships between viral loads and clinical variables were evaluated using Spearman's correlation and point-biserial correlation.
A p-value of <0.05 was maintained as the threshold of statistical significance.
详细描述
Detailed Description Patients with inflammatory bowel disease (IBD) are at increased risk of infections and reactivation of latent pathogens. This study aimed to evaluate Cytomegalovirus (CMV) viral load in the intestinal tissues of pediatric patients, including both IBD and non-IBD groups, and to identify factors associated with increased CMV detection. The study also aimed to establish potential threshold values for CMV viral load in the colonic mucosa, primarily in Ulcerative Colitis (UC) patients, where CMV colitis is more frequently observed, as well as in patients with other intestinal diseases. Finally, diagnostic methods-including histopathology, serum CMV-Polymerase Chain Reaction (CMV-PCR), and tissue CMV-PCR-were compared in terms of diagnostic value.
This prospective study was conducted on pediatric patients who underwent colonoscopy at the Pediatric Gastroenterology Endoscopy Unit between May 2022 and March 2024.
During colonoscopy, two biopsy samples were obtained from the rectal mucosa. In patients with rectal ulcers, biopsies were obtained from the ulcerated area; in patients without ulcers, biopsies were taken from macroscopically normal tissue. Two biopsies were collected from each site: one for tissue CMV-PCR analysis and one for histopathological examination. To prevent contamination, separate forceps were used for samples intended for CMV-PCR.
Concurrent blood samples were analyzed by CMV-PCR and serology (anti-CMV IgG and anti-CMV IgM). CMV IgG avidity was assessed in IgM-positive patients. CMV colitis was diagnosed based on clinical and histopathological findings (IHC and HE staining), along with serum CMV-PCR positivity ≥1000 copies/mL.
2.1. CMV-Deoxyribonucleic Acid (DNA) Extraction from Biopsy Samples Deoxyribonucleic Acid extraction from biopsy samples was performed using the QIAamp DNA Mini Kit [Qiagen, Düsseldorf, Germany] following the manufacturer's instructions. Approximately 25 mg of biopsy tissue was combined with 180 µL of tissue lysis buffer and 20 µL of proteinase K and then vortexed. Samples were incubated overnight at 56°C until the tissue was completely digested. Subsequently, 200 µL of tissue lysis buffer was added, and the samples were incubated at 70°C for 10 minutes. After adding 200 µL ethanol, the mixture was vortexed, centrifuged, and transferred to spin columns. Following washing and centrifugation, DNA was eluted according to the manufacturer's protocol.
研究设计
- 研究类型
- Observational
- 观察模型
- Cohort
- 时间视角
- Prospective
入排标准
- 年龄范围
- 1 Month 至 18 Years（Child, Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •Pediatric patients undergoing colonoscopy for any clinical indication were eligible for inclusion after obtaining written informed consent.
排除标准
- •Patients with insufficient bowel preparation for a high-quality colonoscopic examination.
- •Biopsy Contraindications: Individuals with medical conditions precluding biopsy, such as bleeding diathesis.
- •Non-Participation: Patients or guardians who declined to participate in the study.
- •Children who were undergoing treatment for CMV disease
- •Children who were receiving antiviral therapy (e.g. acyclovir)
研究组 & 干预措施
The patients who underwent colonoscopy at the Pediatric Gastroenterology Department.
Biopsy specimens were obtained from the intestinal mucosa of pediatric patients who underwent colonoscopy for any clinical indication within the specified period.
干预措施: Colonoscopy after bowel cleansing (Diagnostic Test)
结局指标
主要结局
Tissue and serum CMV Viral Load
时间窗: 1,5 year
Cytomegalovirus Polymerase chain reaction levels were detected in both blood and tissue samples across the entire patient population
次要结局
未报告次要终点
研究者
Ödül EGRİTAS
Professor Doctor
Gazi University