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 establish a multiplex polymerase chain reaction (M-PCR) assay for simultaneous detection of pathogens causing genital ulcer disease (GUD).

METHODSBased on the gene-specific region of the following pathogens: Chlamydia trachomatis omp1/ompb, herpes simplex virus (HSV) DNA polymerase, Treponema pallidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen.

RESULTSOf the 51 clinical samples, M-PCR showed slightly higher positive rate (47.1%) of HSV than cell culture (23.6%). Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% (10/51) and 15.7% (8/51), respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture.

CONCLUSIONThis primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.

Chlamydia trachomatis ; genetics ; isolation & purification ; DNA Primers ; Haemophilus ducreyi ; genetics ; isolation & purification ; Herpesvirus 1, Human ; genetics ; isolation & purification ; Herpesvirus 2, Human ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; Sexually Transmitted Diseases ; complications ; microbiology ; virology ; Treponema pallidum ; genetics ; isolation & purification ; Ulcer ; complications ; microbiology ; virology

OBJECTIVETo survey the prevalence and risk factors of HSV-2 among Chinese and Vietnamese female sex workers (FSW) in the border county of Hekou, Yunnan Province, China.

METHODSA cross-sectional survey was conducted on demographics, sexual behavior, medical history, and drug use among FSWs. Laboratory samples were obtained to test for HSV-2 and other STIs such as HIV, Syphilis, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Bacterial vaginosis, and Yeast infections. Cervicitis and genital warts were also diagnosed.

RESULTSOf the 345 FSWs who participated in this study, 112 (32.5%) were ethnic Chinese and 233 (67.5) were Vietnamese. Among FSWs in Hekou, the prevalence rates were 58.3% for HSV-2, 5.5% for HIV, and 4.1% for bacterial vaginosis (BV). Age<21 (OR: 0.5; 95% CI: 0.3, 0.8), duration of commercial sex work≤3 months (OR: 0.5; 95% CI: 0.3, 0.8), oral and vaginal sex with the last client (as opposed to only vaginal sex) (OR: 1.6; 95% CI: 1.0, 2.7), HIV (OR: 11.4; 95% CI: 1.5, 87.2), and bacterial vaginosis (BV) (OR: 5.6; 95% CI: 1.2, 26.9) were significantly correlated with HSV-2 infection.

CONCLUSIONMultivariate analysis showed that several factors were significantly correlated with the high prevalence of HSV-2 in FSWs in the border area between China and Vietnam. Further studies and interventions are needed for HSV-2 epidemiology in the border area.

Adolescent ; China ; epidemiology ; Cross-Sectional Studies ; Female ; Herpes Genitalis ; epidemiology ; Herpesvirus 2, Human ; isolation & purification ; Humans ; Risk Factors ; Sex Workers ; Vietnam ; epidemiology ; Young Adult

OBJECTIVETo study the role of pathogenic microorganisms commonly associated with chronic eye disease, including Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae, herpes simplex virus (HSV) type 1 and type 2, and adenovirus type 8 and type 19, in the development of primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma in Chinese patients.

METHODSSixty-eight archival cases of primary ocular adnexal lymphoproliferative lesions, including 38 cases of MALT lymphoma, 3 cases of non-MALT lymphoma and 27 cases of chronic inflammation, were enrolled into the study. DNA was extracted from the paraffin-embedded tissue samples. The presence of DNA of C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 were analyzed by multiplex touchdown enzyme time-release polymerase chain reaction (TETR-PCR).

RESULTSAll of the specimens yielded PCR products of over 100 base pairs and were thus suitable for TETR-PCR screening of infectious agents. The prevalence of DNA of C. psittaci, C. trachomatis and adenovirus type 19 were 0 in MALT lymphoma, non-MALT lymphoma and chronic inflammation. There were 2 cases positive for C. pneumoniae DNA, amongst the 38 cases of MALT lymphoma studied (5.3%, 2/38). HSV type 1, HSV type 2 and adenovirus type 8 DNA was found in each of the 3 patients with chronic inflammation.

CONCLUSIONThe study indicates that C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 probably play little role in the pathogenesis of ocular adnexal MALT lymphoma in Chinese patients.

Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; isolation & purification ; Chlamydia Infections ; microbiology ; Chlamydia trachomatis ; genetics ; isolation & purification ; Chlamydophila Infections ; microbiology ; Chlamydophila pneumoniae ; genetics ; isolation & purification ; Chlamydophila psittaci ; genetics ; isolation & purification ; DNA, Bacterial ; analysis ; DNA, Viral ; analysis ; Eye Infections ; microbiology ; virology ; Eye Neoplasms ; microbiology ; virology ; Herpes Simplex ; virology ; Herpesvirus 1, Human ; genetics ; isolation & purification ; Herpesvirus 2, Human ; genetics ; isolation & purification ; Humans ; Lymphoma, B-Cell, Marginal Zone ; microbiology ; virology ; Psittacosis ; microbiology

INTRODUCTIONHerpes simplex virus type 2 (HSV-2) is the most common cause of genital herpes. Glycoprotein G (gG) is a prototype antigen for type-specific serodiagnosis distinguishing between HSV type 1 (HSV-1) and HSV-2 infections. As immunological diagnosis kits for accurate differentiation between HSV-1 and HSV-2 antibodies can be expensive, there is a need to develop a convenient, sensitive, specific and cost-effective serodiagnostic kit.

METHODSWe successfully expressed a fragment of gG comprising residues 321-580 of HSV-2 with histidine tag (gG(321-580His)) in a Bac-to-Bac baculovirus expression system, which had an antigenicity similar to its native counterpart. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using gG(321-580His) as the diagnostic antigen and evaluated by comparison with a commercial HerpeSelect 2 ELISA immunoglobulin G kit as reference.

RESULTSIn testing 318 field serum samples, the diagnostic relative sensitivity and specificity of the developed gG(321-580His)-ELISA test in qualitative comparison with the commercial kit were 93.81% and 96.74%, respectively, and the accuracy was 94.65%.

CONCLUSIONThe study indicates that gG(321-580His) has a high diagnostic potential for HSV-2 virus serodiagnosis in humans.

Adult ; Antibodies, Viral ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Herpes Genitalis ; diagnosis ; virology ; Herpes Simplex ; diagnosis ; virology ; Herpesvirus 1, Human ; isolation & purification ; Herpesvirus 2, Human ; isolation & purification ; Humans ; Immunoglobulin G ; chemistry ; Male ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Reference Values ; Reproducibility of Results ; Sensitivity and Specificity ; Serologic Tests ; methods

OBJECTIVETo analyze the prevalence rates and associated risk factors on HIV, syphilis, herpes simplex virus-2 (HSV-2) among female sex workers (FSWs) in Jiaozhou, Shandong province.

METHODSThrough convenient sampling, an anonymous questionnaire survey was conducted on female sex workers to collect related information. Blood specimens were drawn for serological tests on HIV, syphilis and HSV-2 antibodies, respectively. Patients with positive results from both treponema pallidum and HSV-2 tested by ELISA method, were defined as being superinfected.

RESULTSA total of 460 FSWs were recruited in this study. The prevalence rates of HIV, syphilis, HSV-2, and syphilis/HSV-2 superinfection were 0.22% , 5.9% , 43.0% , and 11.7% , respectively. Among the methamphetamine users, the prevalence rates of syphilis, HSV-2, and syphilis/HSV-2 superinfection were 12.4%, 55.2%, and 22.9%, respectively. Results from multivariate analysis showed that factors that significantly associated with syphilis infection would include: methamphetamine use (OR = 3.61, 95% CI:1.62-8.06), having first commercial sex intercourse at under 20 years of age (OR = 2.80, 95% CI:1.15-6.85), more than 2 establishments that the FSWs worked in the last month (OR = 4.37, 95%CI:0.83-22.83). Factors associated with HSV-2 infection were methamphetamine use (OR = 2.30, 95%CI:1.43-3.70), having first commercial sex intercourse at under 20 years of age (OR = 1.77, 95% CI:1.16-2.69), working at low-end establishments (OR = 2.61, 95%CI:1.66-4.10), working at the local sex-work venues for more than one year (OR = 2.01, 95% CI:1.28-3.14), with low education backgrounds (OR = 2.27, 95% CI:1.18-4.36), using methamphetamine (OR = 3.95, 95% CI:2.09-7.44), low education background (OR = 2.43, 95% CI: 1.10-5.36), 21-30 year-olds (OR = 2.95, 95%CI:1.08-8.03), older than 31 years of age (OR = 7.05, 95% CI:2.48-20.01) etc., were independent risk factors associated with the superinfection of syphilis and HSV-2.

CONCLUSIONThe prevalence rates of syphilis, HSV-2 and syphilis/HSV-2 superinfection were relatively high among FSWs in Jiaozhou, especially among the FSWs who were methamphetamine users.

Age Distribution ; Amphetamine-Related Disorders ; epidemiology ; China ; epidemiology ; Educational Status ; Female ; HIV Infections ; epidemiology ; HIV Seropositivity ; Herpes Genitalis ; epidemiology ; Herpesvirus 2, Human ; isolation & purification ; Humans ; Methamphetamine ; administration & dosage ; Prevalence ; Risk Factors ; Serologic Tests ; Sex Work ; Sex Workers ; psychology ; statistics & numerical data ; Superinfection ; epidemiology ; Syphilis ; epidemiology ; Treponema pallidum ; isolation & purification

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

Objective: This study aimed to understand the herpes simplex virus-2 (HSV-2) infection and related factors among female drug abusers in the women's compulsory drug rehabilitation center of Shandong province and to provide reference for the prevention and control of HSV-2 in these settings. Methods: We screened all of 451 female drug abusers in the women's compulsory drug rehabilitation centers in of Shandong province and conducted a study using both questionnaire investigation and serological tests for HSV-2, HIV and syphilis. We also used EpiData 3.1 software to establish a database and SPSS 20.0 software to conduct the χ(2) test and multivariate logistic regression analysis. Results: A total of 451 female drug abusers were under study. We noticed that the rates for HSV-2 infection, HIV infection and syphilis infection appeared as 72.1% (325/451), 2.2% (10/451) and 33.5% (151/451) respectively. Results from univariate analysis showed that factors as: awareness on AIDS, having temporary sex partner after using the drug, having multiple sex partners after using the drug, providing commercial services or having temporary sex practice before being detained, with syphilis infection etc., were associated with HSV-2 infection. Data from the multivariate analysis showed that the OR (95%CI) value of HSV-2 infection was 2.90 (1.19-7.06) for those who providing commercial service, when comparing to those who did not. Compared to those who did not suffer from syphilis infection, the OR (95%CI) value of HSV-2 infection for those with syphilis infection was 2.75 (1.63-4.63). Conclusions: The rate of HSV-2 infection was high in the women's compulsory drug rehabilitation center of Shandong province. We should enhance measures and promote condom use to prevent from HSV-2 and other sexually transmitted diseases among them.
Drug Users ; Female ; HIV Infections/epidemiology* ; Herpes Genitalis/epidemiology* ; Herpes Simplex/epidemiology* ; Herpesvirus 2, Human/isolation & purification* ; Humans ; Opiate Substitution Treatment ; Prevalence ; Risk Factors ; Serologic Tests/methods* ; Sexual Behavior ; Sexual Partners ; Substance Abuse Treatment Centers ; Substance-Related Disorders/rehabilitation* ; Syphilis/epidemiology*