BACKGROUND: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are increasingly important in immunocompromised patients. Nucleic acid extraction methods could affect the results of viral nucleic acid amplification tests. We compared two automated nucleic acid extraction systems for detecting CMV and EBV using real-time PCR assays. METHODS: One hundred and fifty-three whole blood (WB) samples were tested for CMV detection, and 117 WB samples were tested for EBV detection. Viral nucleic acid was extracted in parallel by using QIAsymphony RGQ and QIAcube (Qiagen GmbH, Germany), and real-time PCR assays for CMV and EBV were performed with a Rotor-Gene Q real-time PCR cycler (Qiagen). Detection rates for CMV and EBV were compared, and agreements between the two systems were analyzed. RESULTS: The detection rate of CMV and EBV differed significantly between the QIAsymphony RGQ and QIAcube systems (CMV, 59.5% [91/153] vs 43.8% [67/153], P=0.0005; EBV, 59.0% [69/117] vs 42.7% [50/117], P=0.0008). The two systems showed moderate agreement for CMV and EBV detection (kappa=0.43 and 0.52, respectively). QIAsymphony RGQ showed a negligible correlation with QIAcube for quantitative EBV detection. QIAcube exhibited EBV PCR inhibition in 23.9% (28/117) of samples. CONCLUSIONS: Automated nucleic acid extraction systems have different performances and significantly affect the detection of viral pathogens. The QIAsymphony RGQ system appears to be superior to the QIAcube system for detecting CMV and EBV. A suitable sample preparation system should be considered for optimized nucleic acid amplification in clinical laboratories.
Automation ; Cytomegalovirus/*genetics/isolation & purification ; Cytomegalovirus Infections/diagnosis/*virology ; DNA, Viral/*blood/isolation & purification/metabolism ; Herpesvirus 4, Human/*genetics/isolation & purification ; Humans ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction

Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10(6) IU/mL (R2 ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values <1,000 copies/mL, 100% of the samples had a variation <0.7 log10 copies/mL; >1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had <0.5 log10 copies/mL. The Real-Q assay is useful for quantifying CMV in WB with the two real-time PCR platforms.
Cytomegalovirus/*genetics/isolation & purification ; Cytomegalovirus Infections/diagnosis/virology ; DNA, Viral/*blood/metabolism ; Humans ; Limit of Detection ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the incidence, clinical features, and treatment of perinatal cytomegalovirus (CMV) infection, as well as the factors affecting the therapeutic effect of ganciclovir.

METHODSThe clinical data of 237 infants who were hospitalized and diagnosed with perinatal CMV infection from 2008 to 2012 were retrospectively analyzed.

RESULTSThe clinical features of infants with perinatal CMV infection and the proportion of such infants in all hospitalized infants showed no significant differences across the five years. In most infants, two or more systems were involved, and CMV hepatitis plus CMV pneumonia was most common (43.1%). The results of pathogen detection showed that the percentage of the infants with positive blood CMV-IgM and blood/urine CMV-DNA was 3.8%, while 90.3% of all infants had positive blood CMV-IgM alone and 5.9% had positive blood/urine CMV-DNA alone. A total of 197 infants were treated with ganciclovir, and the cure rate was 88.3%. An abnormal history of pregnancy (OR=6.191, 95% CI: 1.597-24.002) and liver involvement before medication (OR=3.705, 95% CI: 1.537-8.931) were the independent risk factors affecting the therapeutic effect of ganciclovir in infants with perinatal CMV infection.

CONCLUSIONSThe epidemiological characteristics of perinatal CMV infection have remained generally stable for the last 5 years. CMV often involves several organs or systems, especially the liver and lung. Ganciclovir has a significant efficacy in the treatment of perinatal CMV infection, and an abnormal history of pregnancy and liver involvement before medication can increase the risk of ganciclovir resistance in infants with perinatal CMV infection.

Antiviral Agents ; therapeutic use ; Cytomegalovirus ; isolation & purification ; physiology ; Cytomegalovirus Infections ; drug therapy ; epidemiology ; virology ; Female ; Ganciclovir ; therapeutic use ; Humans ; Infant ; Infant, Newborn ; Infant, Newborn, Diseases ; drug therapy ; epidemiology ; virology ; Liver ; virology ; Male ; Retrospective Studies

OBJECTIVETo investigate the clinical and pathologic features of gastrointestinal cytomegalovirus (CMV) disease, and the histological detection of CMV.

METHODSForty-one gastrointestinal tissues were obtained from 35 patients suspected for gastrointestinal CMV disease. The pathologic changes of these specimens were evaluated by immunohistochemistry and in situ hybridization for CMV, and these were compared to serologic detection methods.

RESULTSCMV was detected in 23/41 tissues. There were 32 serologic CMV DNA tests at the time of biopsies or operations. Compared with histological detection, the sensitivity and specificity of serologic CMV DNA tests were 73.7% and 69.2% respectively.

CONCLUSIONSGastrointestinal histological detection of CMV has important clinical application. HE staining combined with immunohistochemistry and/or in situ hybridization detection is a reliable method to detect CMV.

Biopsy ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; Gastrointestinal Diseases ; virology ; Humans ; Immunohistochemistry ; In Situ Hybridization

PURPOSE: Extremely low birth weight infants (ELBWIs) have a high risk of acquiring cytomegalovirus (CMV) infection via breast milk and consequently developing serious symptoms. We evaluated whether freeze-thawing or pasteurization could prevent postnatal CMV infection transmitted through breast milk in ELBWIs. MATERIALS AND METHODS: Medical records of 385 ELBWIs with whole milk feeding, and freeze-thawed or pasteurized breast milk feeding were reviewed retrospectively. Postnatally acquired CMV infection was defined as an initial negative and a subsequent positive on follow-up urine CMV DNA polymerase chain reaction screening tests. The incidence, clinical characteristics, symptoms, sequelae, and long-term outcome at corrected age [(CA): 2 years of CMV infection] were analyzed. RESULTS: While no infant developed CMV infection with whole milk (0/22) or pasteurized breast milk (0/62) feeding, postnatal CMV infection was diagnosed in 8% (27/301) of ELBWIs who were fed freeze-thawed breast milk. Gestational age in the CMV group was significantly lower than the control group. In 82% (22/27) of cases, CMV infection was symptomatic and was associated with increased ventilator days and > or =moderate bronchopulmonary dysplasia (BPD). Neurodevelopmental outcome and growth status at CA 2 years were not different between the study groups. Lower gestational age and freeze-thawed breast milk feeding >60% of total oral intake during the first 8 postnatal weeks were independent risk factors for acquiring postnatal CMV infection. BPD (> or =moderate) was the only significant adverse outcome associated with this CMV infection. CONCLUSION: Pasteurization but not freeze-thawing of breast milk eradicated the postnatal acquisition of CMV infection through breast milk.
Adult ; Breast Feeding ; Bronchopulmonary Dysplasia ; Cytomegalovirus/*isolation & purification ; Cytomegalovirus Infections/epidemiology/prevention & control/*transmission ; Female ; Gestational Age ; Humans ; Incidence ; Infant ; *Infant, Extremely Low Birth Weight ; Infant, Newborn ; Infectious Disease Transmission, Vertical/*prevention & control ; Male ; Milk, Human/chemistry/*virology ; Polymerase Chain Reaction ; Pregnancy ; Pregnancy Complications, Infectious/diagnosis ; Retrospective Studies ; Risk Factors

OBJECTIVETo monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.

METHODSHCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.

RESULTSHCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.

CONCLUSIONSPhenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.

Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; genetics ; isolation & purification ; Drug Resistance, Viral ; genetics ; Ganciclovir ; pharmacology ; Genes, Viral ; Genotype ; Hematopoietic Stem Cell Transplantation ; Humans ; Mutation ; Phosphotransferases (Alcohol Group Acceptor) ; genetics

OBJECTIVETo investigate the associations between Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) infections and the methylation levels of PTEN and hTERT genes and explore their roles in children with acute lymphoblastic leukemia (ALL).

METHODSBlood samples from 100 children with newly diagnosed acute lymphoblastic leukemia were centrifuged for serological detection of EBV and HCMV, and the patients were divided accordingly into EBV-infected group (n=20), HCMV-infected group (n=14), EBV and HCMV co-infected group (n=41), and non-infected group (control group, n=15). DNA was extracted from peripheral blood mononuclear cells (PBMCs) and modified with bisulfite ammonia sodium. The methylation levels of the promoters of PTEN and hTERT genes were detected with methylation-specific polymerase chain reaction (MS-PCR).

RESULTSCompared with those in non-infected group and EBV- or HCMV-infected group, the methylation levels of PTEN gene in the co-infected group were significantly decreased (P<0.05) while the methylation levels of hTERT gene significantly increased (P<0.05).

CONCLUSIONIn children with acute lymphoblastic leukemia, EBV and HCMV co-infection cause changes in the methylation levels of PTEN and hTERT. These results may be associated with epigenetic changes caused by viral infections, and further studies are needed to further verify this hypothesis.

Child ; Coinfection ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; DNA Methylation ; Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; isolation & purification ; Humans ; PTEN Phosphohydrolase ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; genetics ; virology ; Promoter Regions, Genetic ; Telomerase ; genetics ; metabolism

No abstract available.
Antiviral Agents/therapeutic use ; Biopsy ; Chest Pain/diagnosis/*etiology ; Cytomegalovirus/*isolation & purification ; Cytomegalovirus Infections/diagnosis/drug therapy/*virology ; Esophagitis/diagnosis/drug therapy/*virology ; Esophagoscopy ; Ganciclovir/therapeutic use ; Humans ; Kidney Transplantation/*adverse effects ; Male ; Middle Aged ; Opportunistic Infections/diagnosis/drug therapy/*virology ; Treatment Outcome

INTRODUCTIONChronic bacterial, viral and parasitic infections contribute to the morbidity and mortality associated with human immunodeficiency virus (HIV) infection. This study investigated risk factors and time-trends of the seroprevalence of cytomegalovirus (CMV), toxoplasmosis and hepatitis A total antibody; and co-infection with syphilis, hepatitis B and hepatitis C among newly diagnosed HIV individuals in Singapore.

MATERIALS AND METHODSThis was a cross-sectional study. A random sample of 50% of HIV infected patients who visited the Communicable Disease Centre (CDC), Singapore for first-time care from January 2006 to December 2011 were analysed.

RESULTSAmong the 793 study subjects, 93.4% were male; 77.9% of them were of Chinese ethnicity; mean age at HIV diagnosis was 41.4 years; and the mean baseline CD4+ T-cell count was 222 cells/mm³. The prevalence of sero-reactivity for CMV was 96.8%; hepatitis A: 40.9%; and toxoplasmosis: 23.7%. Co-infection with syphilis was identified in 12.3%; hepatitis B: 8.1%; and hepatitis C: 2%. Among those co-infected with hepatitis C, 73.3% of them were intravenous drug user (IVDU). Syphilis co-infection was significantly more common among men who have sex with men (MSM) (multivariate OR: 2.53, 95% CI, 1.31 to 4.90, P = 0.006).

CONCLUSIONThis study described the baseline rates of HIV co-infection with syphilis, hepatitis B and C in Singapore, and sero-reactivity to CMV, toxoplasmosis and hepatitis A. The increased rates compared to the general population may have important consequences for disease progression, response to antiretroviral treatment and long-term general health.

Adult ; Coinfection ; epidemiology ; Cross-Sectional Studies ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; blood ; epidemiology ; Female ; HIV Infections ; epidemiology ; Hepacivirus ; isolation & purification ; Hepatitis, Viral, Human ; blood ; epidemiology ; Humans ; Male ; Odds Ratio ; Risk Factors ; Seroepidemiologic Studies ; Singapore ; epidemiology ; Syphilis ; blood ; epidemiology ; Time Factors ; Toxoplasmosis ; blood ; epidemiology

Autoimmune Diseases ; pathology ; Biopsy ; Cytomegalovirus Infections ; pathology ; Gastric Mucosa ; pathology ; virology ; Gastritis ; pathology ; virology ; Helicobacter Infections ; pathology ; Helicobacter heilmannii ; isolation & purification ; Helicobacter pylori ; isolation & purification ; Humans