The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.
Cytomegalovirus/classification/*isolation & purification ; Herpesvirus 1, Human/classification/*isolation & purification ; Herpesvirus 2, Human/classification/*isolation & purification ; Herpesvirus 4, Human/classification/*isolation & purification ; Human ; *Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Support, Non-U.S. Gov't

OBJECTIVETo provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.

METHODSViruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.

RESULTSNGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.

CONCLUSIONThe improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Clinical Laboratory Techniques ; DNA Barcoding, Taxonomic ; DNA Primers ; Enterovirus ; classification ; genetics ; isolation & purification ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Influenza B virus ; genetics ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods ; Sequence Analysis, RNA ; methods

Adolescent ; Adult ; Aged ; Child ; Diagnosis, Differential ; Female ; Herpesvirus 4, Human ; isolation & purification ; Hodgkin Disease ; classification ; metabolism ; pathology ; virology ; Humans ; Lymphocytes ; pathology ; Male ; Middle Aged ; PAX5 Transcription Factor ; metabolism ; RNA, Viral ; metabolism ; Trans-Activators ; metabolism ; Young Adult

OBJECTIVE: To investigate the prevalence of human papillomavirus in nasopharyngeal carcinomas of Fujian province in China. METHOD: Samples from 70 patients with NPC and 25 controls. All samples were detected HPV DNA by polymerase chain reaction (PCR) suing GP5+/6+ and MY09/11 primers and genotyped by surface plasmon resonance (SPR) and HPV 16/18 E6 and LMP-1 using immunohistochemistry and EBER using in situ hybridization. RESULT: Only 2 cases of 70 patients were showed evidence of HPV DNA by PCR, the 2 HPV positive cases subtype HPV-70 and HPV-18 were genotyped by SPR, both the 2 HPV positive cases are non-keratinizing carcinomas (the HPV-70 positive one is differentiated and the HPV-18 positive one is undifferentiated), both the 2 HPV positive cases do not show any evidence of EBV. Data showed that 57 of 70 NPC detected EBER positive, but only 25 out of 70 NPC samples were detected LMP-1 positive. CONCLUSION Our study showed a low prevalence of human papillomavirus in NPC patients of Fujian province in Southern China, there is no evidence about HPV and EBV co-infection.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Carcinoma ; China ; epidemiology ; DNA, Viral ; isolation & purification ; Epstein-Barr Virus Infections ; epidemiology ; Female ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; epidemiology ; virology ; Papillomaviridae ; classification ; isolation & purification ; Papillomavirus Infections ; epidemiology ; Young Adult

OBJECTIVETo investigate the differences between primary mediastinal B-cell lymphoma (PMBCL) and non-mediastinal conventional diffuse large B-cell common lymphoma (DLBCL) in immunoglobulin gene rearrangement and EB virus infections.

METHODSTwenty cases of PMBCL and 30 cases of non-mediastinal DLBCL were collected from September, 2000 to May, 2011. Pathological data were retrospectively analysed. Immunoglobulin heavy chain and light chain gene rearrangements and EBER in-situ hybridization were performed.

RESULTSSix of 20 cases of PMBCL showed monoclonal gene rearrangement, all of which were weakly detected. Twenty-seven of 30 cases of ordinary diffuse large B-cell lymphoma showed monoclonal gene rearrangement, which were strongly detected (90.0%). Only 1 of 20 cases PMBCL and 2 of 30 cases of DLBCL were positive for EBER in-situ hybridization.

CONCLUSIONSThe detection rate of immunoglobulin gene rearrangement is significantly lower in PMBCL than that of non-mediastinal DLBCL. However, EB virus infection rates are very low in both types of lymphomas.

Adult ; Epstein-Barr Virus Infections ; Female ; Gene Rearrangement, B-Lymphocyte ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; In Situ Hybridization ; Lymphoma, B-Cell ; genetics ; virology ; Lymphoma, Large B-Cell, Diffuse ; classification ; genetics ; virology ; Male ; Mediastinal Neoplasms ; genetics ; virology ; Middle Aged ; RNA, Viral ; analysis ; Retrospective Studies ; Young Adult

OBJECTIVETo study the immunophenotype and overall survival of diffuse large B-cell lymphoma (DLBCL) classified according to the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues.

METHODSFive hundred cases of DLBCL were retrospectively analyzed with histologic review, immunohistochemistry, gene rearrangement study, in situ hybridization and fluorescence in situ hybridization. Follow-up data were collected. The overall survival rates of germinal center B-cell (GCB) and non-germinal center B-cell (non-GCB) subtypes, as well as those of DLBCL, not otherwise specified (NOS) and Epstein-Barr virus (EBV)-positive DLBCL of the elderly, were compared.

RESULTSDLBCL-NOS was the commonest subtype which accounted for 77.2% (386/500) of the cases. EBV-positive DLBCL of the elderly, primary DLBCL of central nervous system, primary mediastinal (thymic) large B-cell lymphoma and T cell/histiocyte-rich large B-cell lymphoma accounted for 9.4% (47/500), 4.4% (22/500), 2.8% (14/500) and 2.6% (13/500), respectively. 68.5% (219/320) of DLBCL-NOS belonged to non-GCB subtype. The percentage of GCB subtype and CD5-positive subtype were 28.4% (91/320) and 3.1% (10/320), respectively. Comparison of the overall survival, GCB and non-GCB immunophenotypic groups have no significant difference (P = 0.93). And the same result in which of the EBV-positive DLBCL of the elderly and DLBCL-NOS group, before and after age matched (P = 0.13 and 0.28, respectively). A double-hit lymphoma was found by FISH detection, which presenting as gray zone lymphoma in morphology.

CONCLUSIONSBy using Hans algorithm, GCB and non-GCB subtypes show no significant difference in overall survival. EBV-positive DLBCL of the elderly and DLBCL-NOS also do not have significant difference in overall survival. Fluorescence in situ hybridization technique is helpful in identification of DLBCL with rare phenotypes.

Aged ; Burkitt Lymphoma ; metabolism ; pathology ; CD5 Antigens ; metabolism ; Epstein-Barr Virus Infections ; pathology ; Follow-Up Studies ; Genes, Immunoglobulin Heavy Chain ; Genes, bcl-2 ; Germinal Center ; pathology ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Immunophenotyping ; Interferon Regulatory Factors ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; classification ; genetics ; pathology ; Middle Aged ; Neprilysin ; metabolism ; Oncogene Fusion ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; Retrospective Studies ; Survival Rate

Castleman's disease represents an atypical lymphoproliferative disorder, infrequently associated with various immunologic abnormalities or subsequent development of malignancy such as Kaposi sarcoma, malignant lymphoma and plasmacytoma. Its clinicopathologic features depend on various etiologic factors such as Kaposi sarcoma herpesvirus (KSHV), oversecretion of IL-6, adhesion molecule and follicular dendritic cell dysplasia, etc. To investigate the relationship of Castleman's disease (CD) and the above factors, we reviewed 22 cases of CD. Four cases of KSHV positive CD were detected, all multicentric, plasma cell type, and these cases displayed prominent vascular proliferation, characteristic 'Kaposi-like lesion'. IL-6 and CD54 positive mononuclear cells were scattered in interfollicular areas of KSHV positive cases. Follicular dendritic cell hyperplasia, vascular proliferation, expression of IL-6 and CD54 did not show any significant difference between solitary vs multicentric type, and plasma cell type vs hyaline vascular type. Our study suggests that KSHV positive CD reveals unique pathologic features, and the probable relationship of KSHV and IL-6 and CD54 is discussed.
Adolescence ; Adult ; Biological Markers ; Dendritic Cells, Follicular/pathology ; Epstein-Barr Virus Infections/virology ; Epstein-Barr Virus Infections/epidemiology ; Female ; Germinal Center/pathology ; Giant Lymph Node Hyperplasia/virology ; Giant Lymph Node Hyperplasia/pathology+ACo- ; Giant Lymph Node Hyperplasia/epidemiology ; Giant Lymph Node Hyperplasia/classification ; Herpesviridae Infections/virology ; Herpesviridae Infections/epidemiology ; Herpesvirus 4, Human/isolation +ACY- purification ; Herpesvirus, Kaposi Sarcoma-Associated/isolation +ACY- purification ; Human ; Hyperplasia ; Intercellular Adhesion Molecule-1/analysis ; Interleukin-6/analysis ; Korea/epidemiology ; Lymph Nodes/virology ; Lymph Nodes/pathology ; Lymph Nodes/chemistry ; Male ; Middle Age ; Neovascularization, Pathologic ; Receptors, Complement 3d/analysis ; Retrospective Studies ; Tumor Virus Infections/virology ; Tumor Virus Infections/epidemiology