Tissue Cytomegalovirus Viral Load and Associated Factors in Pediatric Patients Undergoing Colonoscopy

Investigation of Tissue Cytomegalovirus (CMV) Status Via Polymerase Chain Reaction and Serological Tests in Patients Undergoing Colonoscopy and Intestinal Biopsy

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.

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.

2.2. CMV-DNA Extraction from Plasma Samples Deoxyribonucleic Acid extraction from plasma samples was performed using the QIAsymphony DSP Virus/Pathogen Midi Kit [QIAGEN, Germany] on the QIAsymphony SP instrument [QIAGEN, Germany] according to the manufacturer's guidelines. An internal control was included in each sample to assess the extraction efficiency.

2.3. CMV-DNA Amplification Amplification of the extracted CMV DNA was performed using an Artus CMV QS-RGQ Kit [Qiagen, Germany] on a Rotor-Gene Q instrument [Qiagen, Germany]. This kit enables the specific amplification of a 105-bp region of the CMV genome. For each patient, a 25 µL master mix and 5 µL magnesium solution were prepared, and 30 µL of the mixture was transferred into amplification tubes and combined with patient-derived isolates. The samples were loaded onto a Rotor-Gene Q instrument. Each run included four quantification standards and a negative control to detect potential contamination. Viral loads in the blood and tissue samples were reported as copies/mL and copies/mg, respectively. The analytical sensitivity of the assay was 42.5 copies/mL, with a linear range of 79.4 to 1×10⁸ copies/mL.

2.4. Histopathological Evaluation Four-micrometer-thick sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged slides. CMV expression [Cell Marque, clone 8B1.2 1G5.2 & 2D4.2, 1:500 dilution] was assessed using the Ultraview Universal DAB Detection Kit through a streptavidin-biotin triple indirect immunoperoxidase method on the automated Ventana Benchmark XT platform. Colon mucosa, which is known to be CMV-positive, served as a positive control. Nuclear staining of cells was considered indicative of CMV expression, and biopsies displaying this expression were interpreted as positive.

2.5. StatisticalAnalysis All collected data were recorded in the study form and analyzed using IBM SPSS Statistics version 20.0 [Chicago, IL, USA]. The normality of continuous and discrete variables was assessed using the Shapiro-Wilk test, histograms, and Q-Q plots. Descriptive statistics were presented as mean ± standard deviation [SD] or median [interquartile range, IQR] for continuous variables and as frequency [percentage] for categorical variables. Categorical variables were compared using the Chi-square test, whereas continuous variables were evaluated using Student's t-test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. Spearman's correlation analysis was applied for correlations between non-normally distributed continuous variables, and point-biserial correlation was used for correlations between dichotomous and continuous variables. A significance level of p < 0.05 was considered statistically significant, with a critical alpha value set at 0.05.

Inclusion Criteria

-Pediatric patients undergoing colonoscopy for any clinical indication were eligible for inclusion after obtaining written informed consent.

Exclusion Criteria

• 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)