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

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

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

BACKGROUND/AIMS: Infections are major causes of both early and late death after lung transplantation (LT). The development of prophylaxis strategies has altered the epidemiology of post-LT infections; however, recent epidemiological data are limited. We evaluated infections after LT at our institution by time of occurrence, site of infections, and microbiologic etiologies. METHODS: All consecutive patients undergoing lung or heart-lung transplantation between October 2008 and August 2014 at our institution were enrolled. Cases of infections after LT were initially identified from the prospective registry database, which was followed by a detailed review of the patients' medical records. RESULTS: A total of 108 episodes of post-LT infections (56 bacterial, 43 viral, and nine fungal infections) were observed in 34 LT recipients. Within 1 month after LT, the most common bacterial infections were catheter-related bloodstream infections (42%). Pneumonia was the most common site of bacterial infection in the 2- to 6-month period (28%) and after 6 months (47%). Cytomegalovirus was the most common viral infection within 1 month (75%) and in the 2- to 6-month period (80%). Respiratory viruses were the most common viruses after 6 months (48%). Catheter-related candidemia was the most common fungal infection. Invasive pulmonary aspergillosis developed after 6 months. Survival rates at the first and third years were 79% and 73%, respectively. CONCLUSIONS: Although this study was performed in a single center, we provide valuable and recent detailed epidemiology data for post-LT infections. A further multicenter study is required to properly evaluate the epidemiology of post-LT infections in Korea.
Adult ; Bacterial Infections/diagnosis/*microbiology/mortality ; Catheter-Related Infections/microbiology/virology ; Cytomegalovirus Infections/virology ; Female ; Heart-Lung Transplantation/*adverse effects/mortality ; Humans ; Kaplan-Meier Estimate ; Lung Transplantation/*adverse effects/mortality ; Male ; Medical Records ; Middle Aged ; Mycoses/diagnosis/*microbiology/mortality ; Pneumonia, Bacterial/microbiology ; Registries ; Republic of Korea/epidemiology ; Risk Factors ; Time Factors ; Treatment Outcome ; Virus Diseases/diagnosis/mortality/*virology

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 investigate the clinical predictors of cytomegalovirus (CMV) infection after liver transplantation.

METHODSThe clinical data of 182 patients (146 male and 36 female with a mean age of (50 ± 7) years) receiving liver transplantation in Beijing Chaoyang Hospital between January 2004 and December 2008 were retrospectively analyzed.All patients were divided into two groups, namely the CMV infection group (n=24) and the control group (n=158). Logistic regression was used to identify the predictive factors of postoperative CMV infection.

RESULTSAccording to univariate analysis results, the factors for CMV infection were acute liver failure (P=0.032), MELD score ≥ 30 (P=0.001), liver retransplantation (P=0.002), acute rejection (P=0.000) and delayed graft function (P=0.022). According to multi-analysis results, MELD score ≥ 30 (P=0.037, 95%CI:1.194-271.461) and acute rejection (P=0.033, 95%CI:1.179-51.863) were proved to be independent predictors by multivariate analysis.

CONCLUSIONThe study indicates that MELD score ≥ 30 and acute rejection are the independent predictors of CMV infection.

Adult ; Beijing ; Cytomegalovirus Infections ; diagnosis ; End Stage Liver Disease ; diagnosis ; Female ; Graft Rejection ; Humans ; Liver Transplantation ; adverse effects ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Postoperative Complications ; virology ; Reoperation ; Retrospective Studies ; Severity of Illness Index

To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.
Adolescent ; Adult ; Aged ; Conserved Sequence ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; virology ; DNA, Viral ; blood ; Female ; Humans ; Immunosuppressive Agents ; blood ; Kidney Transplantation ; adverse effects ; Male ; Middle Aged ; Polyomavirus ; genetics ; isolation & purification ; Polyomavirus Infections ; diagnosis ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity ; Species Specificity ; Tacrolimus ; blood ; Time Factors ; Tumor Virus Infections ; diagnosis ; virology ; Viral Load ; Young Adult

OBJECTIVETo evaluate the clinical application value of throat swab nested PCR for detecting active congenital human cytomegalovirus (HCMV) infection in neonates.

METHODSThe throat swabs and umbilical cord blood specimens from 51 neonates were collected for nested PCR assay for HCMV glycoprotein B (gB) gene. Moreover, 18 of them were subjected to a pp65 antigen test.

RESULTSThe sensitivity and specificity of throat swab nested PCR for HCMV gB gene were 67% and 75%, respectively, and the positive and negative predictive values were 57% and 82%, respectively.

CONCLUSIONSThroat swab nested PCR assay for HCMV gB gene is non-invasive, rapid, and highly sensitive for HCMV detection and holds promise as an excellent screening technology for detecting active congenital HCMV infection in neonates.

Cytomegalovirus Infections ; congenital ; diagnosis ; Fetal Blood ; virology ; Humans ; Infant, Newborn ; Phosphoproteins ; blood ; Polymerase Chain Reaction ; methods ; Viral Envelope Proteins ; genetics ; Viral Matrix Proteins ; blood

OBJECTIVETo quantify human cytomegalovirus (HCMV) DNA in the blood and urine of infants of different ages with suspected HCMV infection, and in the breast milk of their mothers, and to evaluate the significance of HCMV DNA detection in the three specimen types in the diagnosis of HCMV infection among infants of different ages.

METHODSA total of 170 infants with suspected HCMV infection were divided into groups A (<28 days; n=43) and B (28 days to 5 months; n=127) according to their ages. Blood and urine were collected from the infants, and breast milk was collected from their mothers. The specimens were examined by fluorescence quantitative PCR for detection of HCMV DNA.

RESULTSIn group A, HCMV DNA detection rates in blood, urine and breast milk were 65.1%, 18.6% and 93.0% respectively. In group B, HCMV DNA detection rates in blood, urine and breast milk were 64.6%, 92.9% and 72.4% respectively. HCMV DNA detection rate in urine in group B was significantly higher than in group A (P<0.01), however, HCMV DNA detection rate in mothers' breast milk in group B was significantly lower than in group A (P<0.01). Among the 82 infants with positive results for blood and urine, the copy number of HCMV DNA in urine was significantly higher than that in blood.

CONCLUSIONSHCMV DNA detection rates in urine and breast milk are different among infants of different ages, so use of suitable specimens according to age is of great significance for improving detection rate.

Animals ; Cytomegalovirus Infections ; diagnosis ; DNA, Viral ; analysis ; blood ; urine ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Milk, Human ; virology ; Pregnancy

Cytomegalovirus (CMV) infections are usually diagnosed in immunocompromised patients. A 74-year-old male without any significant medical history visited our center because of abdominal pain and diarrhea which began about a month ago. Abdominal computed tomography revealed segmental enhanced bowel wall thickening on jejunum and single-balloon enteroscopy showed multiple geographic shaped ulcerations covered with exudates on proximal jejunum. Biopsy samples taken during endoscopic examination demonstrated necrotic fibrinopurulent tissue debris and benign ulcer. Nested-PCR analysis of CMV DNA from jejunal tissue was positive. The patient was finally diagnosed with CMV jejunitis and was treated by intravenous ganciclovir for 14 days after which, abdominal pain and diarrhea improved. Our case shows that CMV jejunitis can occur in an immunocompetent adult as multiple jejunal ulcers which can be diagnosed using a single-balloon enteroscope.
Aged ; Antiviral Agents/therapeutic use ; Cytomegalovirus/genetics/isolation & purification ; Cytomegalovirus Infections/complications/*diagnosis/drug therapy ; DNA, Viral/analysis ; Endoscopy, Gastrointestinal ; Enteritis/*diagnosis/etiology/virology ; Ganciclovir/therapeutic use ; Humans ; Injections, Intravenous ; Jejunal Diseases/*diagnosis/etiology/virology ; Male ; Polymerase Chain Reaction ; Tomography, X-Ray Computed