OBJECTIVE: To investigate the relationship between MICA*008/A5.1 allele and human cytomegalovirus (HCMV) infection in kidney transplanted donees of Hunan Han nationality. METHODS: The MICA*008/A5.1 allele based on 91 kidney transplanted donees and 81 unrelated normal individuals of Han nationality in Hunan Province were analyzed by PCR/SSP assay. At the same time, anti-HCMV antibody IgM was detected in the serum by ELISA method. RESULTS: The positive rate of MICA*008/A5.1 allele was significantly higher in the control group (56.79%) than that in the kidney transplanted donee group (34.07%) (P <0.05). The infection rate of HCMV in those individuals whose genotype was MICA*008/A5.1 (-) was significantly higher than that in the MICA*008/A5.1(+). CONCLUSION The individual whose genotype is MICA*008/A5.1 (+) is not liable to HCMV infection, but the individual whose genotype is MICA*008/A5.1 (-) is liable to HCMV infection.
Alleles ; Antibodies, Viral ; blood ; China ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; genetics ; Female ; Genotype ; Histocompatibility Antigens Class I ; genetics ; Humans ; Immunoglobulin M ; blood ; Kidney Transplantation ; adverse effects ; Male

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 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

BACKGROUNDThe study was designed to investigate the status of molecular epidemiology of HCMV in Urumqi through genetic comparison of clinical isolates.

METHODSDNA sequences of 2.0-2.6 kb were amplified by polymerase chain reaction from three relatively conservative gene regions (DNA polymerase, glycoproteins H, and major immediate-early antigen) of 28 clinical HCMV strains and then were analysed by restriction enzymes.

RESULTSThe restriction patterns of the clinical isolates which did not have relation in epidemiology were greatly different, but the patterns of the clinical isolates related in epidemiology such as strains paired in mother and infant were quite similar. Of eight mother and infant pairs, from whom HCMV were isolated, four pairs showed identity of restriction profiles within each pair for all three amplified regions, four pairs showed differences between mother and infant.

CONCLUSIONThese results confirm the high degree of genetic variability among cytomegalovirus strains in Urumqi. Analysis of PCR-RFLP can indicate transmission of HCMV infection and facilitate its molecular epidemiologic studies.

China ; epidemiology ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; epidemiology ; virology ; DNA-Directed DNA Polymerase ; genetics ; Humans ; Immediate-Early Proteins ; genetics ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Viral Envelope Proteins ; genetics

BACKGROUNDTo study the polymorphism of human cytomegalovirus US28 gene in children and investigate the relationship between the polymorphism and pathogenesis.

METHODSThe FQ-PCR was carried out to determine the DNA quantity of clinical isolate and then the segmental PCR and HMA-SSCP were performed to test the mutation of US28 gene. The typical isolates from different diseases were selected to clone and sequence, then the results were analyzed.

RESULTSThe nucleic acid mutation is frequent among the sequence of US28, those mutations focus on the two ends of US28, but most of them are sense mutation. The important functional groups of US28 are highly conserved. The amino acid mutation of some isolates resulted in the change of secondary structure, but the phylogenetic tree analysis did not show any clear association between the pathogenesis and the distribution of clinical isolates. The comparison of US28 sequences from AIDS patients with the sequences from children in our study showed that both sequences have their own specific high mutation points.

CONCLUSIONThere is polymorphism among the HCMV-US28 gene of clinical isolates from children. There observed no clear relationship was between the pathogenesis and the distribution of clinical isolates.

Base Sequence ; Child ; Congenital Abnormalities ; virology ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; virology ; Genotype ; Humans ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single-Stranded Conformational ; Receptors, Chemokine ; genetics ; Sequence Analysis, DNA ; Viral Proteins ; genetics

BACKGROUNDHuman cytomegalovirus (HCMV) infection is an important infectious agent that results in neonatal disease and congenital deformity. HCMV infection may affect in many organs. The different symptoms and tissue tropism of HCMV infection perhaps resulted from the genetic polymorphism of HCMV. HCMV UL144 open reading frames encode a homologue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection.

METHODSHCMV-UL144 gene was amplified by PCR assay in 65 low-passage clinical isolates and urine from 7 healthy children who were HCMV-DNA positive by quantitative PCR. All the positive PCR products were analyzed by Heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced.

RESULTSFifty-five isolates and 5 urine specimens were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORFs. Comparing UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively.

CONCLUSIONHCMV-UL144 existed in almost all the low passage isolates. HMA-SSCP assay is an easy and effective method to detect the genetype of HCMV-UL144 sequence. The characteristic of sequences in different isolates showed that UL144 gene may play an important role in HCMV infection.

Cytomegalovirus ; classification ; genetics ; isolation & purification ; Cytomegalovirus Infections ; virology ; Genotype ; Humans ; Infant ; Infant, Newborn ; Membrane Glycoproteins ; genetics ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; Viral Proteins ; genetics

OBJECTIVETo explore the effect of CMV gB genotypes on viral load and treatment time in patients with CMV infection after hematopoietic stem cell transplantation (HSCT).

METHODSViral load was detected by real-time (RT) quantitative polymerase chain reaction (PCR) (Q-PCR), CMV gB genotypes by PCR restriction fragment length polymorphism (RFLP) (PCR-RFLP) in 115 patients with CMV infection (CMV-DNA positive) after HSCT during July 2004 and May 2010.

RESULTS(1) The distribution of CMV gB genotypes in HSCT recipients were as following: gB1, 42/115 (36.52%); gB2, 3/115 (2.61%); gB3, 43/115 (37.39%); gB4, 2/115 (1.74%). 20 patients (17.39%) had a combination of 2 different CMV genotypes and 5 patients (4.35%) had a CMV variant that lacked an RsaI digestion site, herein named gB5. (2) The median viral load were 2.7×10(3)(1.81×10(3) ∼ 6.03×10(4)) in gB1, 4.0×10(3) (1.32×10(3) ∼ 6.39×10(4)) in gB3 and 1.2×10(4)(2.28×10(3) ∼ 6.50×10(5)) in mixed gB. There was no statistical difference in viral load between gB1 and gB3 (P > 0.050). There was significantly statistical difference in viral load between single-gB (gB1 or gB3) and mixed-gB (P < 0.05). (3) The median treatment time was 17 days in mixed-gB and 14 days in single-gB. There was significantly statistical difference between two groups (P < 0.05). Conclusion gB genotype may have an impact on CMV DNA load and treatment time in HSCT recipients with CMV infection.

Adolescent ; Adult ; Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; virology ; DNA, Viral ; isolation & purification ; Female ; Genotype ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Viral Envelope Proteins ; genetics ; Viral Load ; Young Adult

BACKGROUNDTo investigate the polymorphism of human cytomegalovirus UL148 gene in low passage clinical isolates and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection.

METHODSPCR was performed to amplify the entire HCMV UL148 gene region of 38 clinical isolates, which had been proven containing detectable HCMV-DNA by using FQ-PCR.PCR amplification products were sequenced directly and the sequence data were analysed.

RESULTSSeventeen of 38 isolates were amplified successfully. By comparison with Toledo sequence, the length of UL148 ORFs in all 17 clinical isolates was similar to that of Toledo. Amino acid variability rate of UL148 protein was 0.3%-2.3%. There were additional or deleted sites of posttranslational modification of UL148 protein in all clinical isolates.

CONCLUSIONAll DNA and deduced amino acid sequences of UL148 gene shared great similarity among HCMV clinical strains regardless of their polymorphism.

Amino Acid Sequence ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; congenital ; virology ; Humans ; Infant ; Infant, Newborn ; Open Reading Frames ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Protein Structure, Secondary ; Viral Proteins ; chemistry ; genetics

OBJECTIVETo evaluate the detection of cytomegalovirus (CMV) by polymerase chain reaction (PCR) for predicting the development of CMV disease.

METHODSOne hundred and thirty one allo-HSCT patients performed in the past 2 years were analyzed retrospectively. PCR-CMV was used to monitor CMV viremia and vireuria once a week after transplantation.

RESULTSIn the dynamic detection, CMV viremia was positive for at least one chance in 89 patients, vireuria did in 99 patients. Thirty-seven patients developed CMV disease with an accumulative incidence of 32.5%. The incidence of CMV disease was 15.6% in plasma CMV-PCR negative group, 31.3% in positive once group, and 47.3% in positive over twice group. There was significant difference among the three groups (P = 0.0126). The incidence of CMV disease was 24.8% in urine CMV-PCR negative group, 43.5% in positive once group, and 33.0% in positive over twice group, being no significant difference among them (P = 0.845). On analysis, viremia could predict the development of CMV disease: the PPV (positive predictive value) is 40.5%, NPV (negative predictive value) is 84.4%, sensitivity is 75.0%, and specificity is 69.2%.

CONCLUSIONSDetected by CMV-PCR, MCV viremia may predict the development of CMV disease, but MCV vireuria cannot.

Adolescent ; Adult ; Child ; Child, Preschool ; Cytomegalovirus ; genetics ; isolation & purification ; Cytomegalovirus Infections ; diagnosis ; etiology ; DNA, Viral ; blood ; urine ; Female ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Retrospective Studies ; Sensitivity and Specificity ; Transplantation, Homologous ; adverse effects