Multicenter comparison of PCR assays for detection of human herpesvirus 8 DNA in semen

Reported prevalences of human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in semen have ranged widely. This is possibly due to differences in assay sensitivity, geographic or population-based differences in the true presence of the virus in semen, and PCR contamination. This study assessed interlaboratory sensitivity and reproducibility in the analysis of blinded experimental panels, each consisting of 48 specimens and being composed of semen specimens from different healthy artificial-insemination donors (n = 30) and human immunodeficiency virus (HIV)-infected patients (n = 7) plus positive (n = 4) and negative (n = 7) controls. The experimental panels analyzed in each laboratory were identical except for being independently coded. Of 10 experiments done in five laboratories, 5 experiments from three laboratories had evidence of PCR contamination; all instances of contamination were in the context of nested PCR procedures. In the experiments with no false-positive results, HHV-8 DNA was detected in three (8%) of the 37 semen specimens (two from artificial-insemination donors and one from an HIV-positive patient) but in only 3 (1.6%) of the 184 PCRs in which these specimens were analyzed. This suggests that HHV-8 DNA is present in semen at concentrations that can be too low to allow its consistent detection. This study emphasizes the importance of performing blinded, multi-institution experiments to provide a coherent basis for comparing results and to motivate standardization of methods.     

HBs-Ag-negative hepatitis in a hemodialysis unit: relation to Epstein-Barr virus.

Eleven of 40 patients in a hemodialysis unit had clinical or biochemical evidence of hepatitis during a five-week period. The clinical disease was mild, being limited solely to dialysis patients. Epidemiologic investigation indicated that the incubation period was between 17 and 35 days and that 10 of 11 patients had been exposed to a single venous-pressure monitor before onset. Dried blood and evidence of blood reflux up the venous-pressure gauge suggested that cross-contamination of the blood of successive patients probably resulted in transmission of disease. No association with the hepatitis B surface antigen or anti-hepatitis B antibody was demonstrated, but 10 of the 11 patients with elevated transaminase levels had evidence of recent exposure, to Epstein-Barr virus as manifested either by Ox-cell hemolysin titers or rises in titers to viral capsid antigen.     

Typing of clinical herpes simplex virus isolates with mouse monoclonal antibodies to herpes simplex virus types 1 and 2: comparison with type-specific rabbit antisera and restriction endonuclease analysis of viral DNA

A total of 122 clinical isolates of herpes simplex virus (HSV) from 107 patients were typed by using an indirect immunoperoxidase technique with commercially available type-specific rabbit antisera, recently developed mouse monoclonal antibodies to HSV types 1 and 2, and restriction endonuclease analysis of viral DNA. With the commercially available type-specific rabbit antisera, 34% of clinical HSV isolates were of indeterminate type; 63% of them were typed as HSV type 1 and 37% as HSV type 2 by using monoclonal antibody and restriction enzyme typing systems. Typing by immunofluorescence assay with the monoclonal antibodies gave identical results to those obtained by restriction enzyme analysis. Simultaneous infection with both HSV types was demonstrated by monoclonal antibody typing in five isolates from three patients. These findings were subsequently confirmed by plaque purification and restriction endonuclease analysis of viral DNA. Monoclonal antibodies were as sensitive as restriction enzyme analysis for the typing of clinical HSV isolates. Because of their simplicity, they are more amenable to use in clinical laboratories than is restriction endonuclease analysis.     

A controlled trial comparing vidarabine with acyclovir in neonatal herpes simplex virus infection. Infectious Diseases Collaborative Antiviral Study Group

BACKGROUND. Despite the use of vidarabine, herpes simplex virus (HSV) infection in neonates continues to be a disease of high morbidity and mortality. We undertook a controlled trial comparing vidarabine with acyclovir for the treatment of neonatal HSV infection. METHODS. Babies less than one month of age with virologically confirmed HSV infection were randomly and blindly assigned to receive either intravenous vidarabine (30 mg per kilogram of body weight per day; n = 95) or acyclovir (30 mg per kilogram per day; n = 107) for 10 days. Actuarial rates of mortality and morbidity among the survivors after one year were compared overall and according to the extent of the disease at entry into the study (infection confined to the skin, eyes, or mouth; encephalitis; or disseminated disease). RESULTS. After adjustment for differences between groups in the extent of disease, there was no difference between vidarabine and acyclovir in either morbidity (P = 0.83) or mortality (P = 0.27). None of the 85 babies with disease confined to the skin, eyes, or mouth died. Of the 31 babies in this group who were treated with vidarabine and followed for a year, 88 percent (22 of 25) were judged to be developing normally after one year, as compared with 98 percent (45 of 46) of the 54 treated with acyclovir (95 percent confidence interval for the difference, -4 to 24). For the 71 babies with encephalitis, mortality was 14 percent with vidarabine (5 of 36) and with acyclovir (5 of 35); of the survivors, 43 percent (13 of 30) and 29 percent (8 of 28), respectively, were developing normally after one year (95 percent confidence interval for the difference, -11 to 39). For the 46 babies with disseminated disease, mortality was 50 percent (14 of 28) with vidarabine and 61 percent (11 of 18) with acyclovir (95 percent confidence interval for the difference, -20 to 40); of the survivors, 58 percent (7 of 12) and 60 percent (3 of 5), respectively, were judged to be developing normally after one year (95 percent confidence interval for the difference, -40 to 50). Both medications were without serious toxic effects. CONCLUSIONS. In this multicenter, randomized, blinded study there were no differences in outcome between vidarabine and acyclovir in the treatment of neonatal HSV infection. The study lacked statistical power to determine whether there were sizable differences within the subgroups of those with localized HSV, encephalitis, or disseminated disease.     

Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions.

Previous studies have shown an association between the approximate titer of herpes simplex virus (HSV) DNA in clinical specimens and the ability to isolate HSV from genital secretions. To control for variance in amplification conditions, we developed a competitive quantitative PCR (QC PCR) for the detection of HSV DNA. The assay accurately measured from 10 to 10(6) copies of HSV DNA. We compared the QC PCR with our previous semiquantitative detection method and found concordance for 61 of 63 positive specimens. We also evaluated the HSV DNA content from individual swabs of genital secretions obtained from individual sites of the genital tract (cervix, vulva, and rectum) with that from one swab with secretions from all three sites. The concordance for detecting HSV DNA was 91%; for only 4 of 143 collection days was there a > 1 log difference between the two collection methods. A single swab with secretions from all three genital sites and evaluated in a QC PCR format can accurately measure the frequency of subclinical and clinical shedding of HSV and the titer of HSV shed from the genital region. Such an approach should be very useful in the evaluation of antiviral chemotherapy for HSV.     

Nonrandom chromosomal changes in untreated retinoblastomas

The karotypic patterns of 15 retinoblastomas were examined. Five tumors were found to have two distinct stem lines and, therefore, the chromosomal patterns of 20 tumor cell lines are reported. Three nonrandom chromosomal changes, namely, a loss of a chromosome #13, the presence of an i(6p), or a trisomy of 1q were observed. The potential importance of these chromosomal changes in tumor development is discussed, particularly the loss of a chromosome #13 or the gain of an i(6p). At least one of the three chromosomal changes was found in 75% of the tumor lines analyzed.     

Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes

Leukotriene D4 (LTD4), the most active spasmogenic leukotriene constituent of the slow reacting substance of anaphylaxis was converted by suspended human polymorphonuclear leucocytes (PMNs) to a single, less polar metabolite which was not further catabolized. This product was identified as leukotriene E4 (LTE4) by its retention time during reverse phase-high performance liquid chromatography (RP-HPLC) and subsequent bioassay on the guinea-pig ileum. LTD4 with a retention time of 21 +/- 1.6 min (mean +/- SD) and a contractile activity of 5.0 +/- 0.4 u./pmol (mean +/- SD) was quantitatively converted extracellularly by PMNs to LTE4 with a retention time of 26 +/- 1.8 min and a contractile activity of 1.2 +/- 0.3 u./pmol. Subcellular fractionations of PMNs revealed the recovered LTD4-to-LTE4 converting activity, termed LTD4 dipeptidase, to be localized only in he granule fraction. There was a time- and calcium-dependent extracellular release of LTD4 dipeptidase in association with lysozyme (r = 0.97, n = 16, P less than 0.001), a constituent of both specific and azurophilic granules, in the absence of release of cytoplasmic lactate dehydrogenase (LDH) and of beta-glucuronidase from the azurophilic granule. Phorbol myristate acetate (PMA), which selectively induces secretion of specific granules, released lysozyme and the LTD4 dipeptidase in a constant dose-dependent manner from PMNs (r = 0.96, n = 8, P less than 0.001). Calcium ionophore A23187 at concentrations less than 10(-7) M stimulated the parallel secretion of LTD4 dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), dipeptidase and lysozyme (r = 0.91, n = 9, P less than 0.005), whereas higher concentrations resulted in secretion of beta-glucuronidase and additional lysozyme without further release of dipeptidase. Thus, human PMNs can convert LTD4 to LTE4, a less vasoactive and spasmogenic leukotriene, via the secretion of a dipeptidase associated with the specific granules.     

Molecular identification of pathogenetic IdLNF+1 autoantibody idiotypes derived from the NZBxSWR F1 model for systemic lupus erythematosus

The acceleration of nephritis in SNF(1) mice by CD4(+) T-cell clones reactive with a nephritogenic idiotype, Id(LN)F(1) [1], as well as the ability of anti-Id(LN)F(1) antisera to down-regulate the production of Id(LN)F(+)(1) immunoglobulin (Ig) in vivo and delay nephritis [2], suggests that dysregulation of this idiotype may contribute to the development of SNF(1) nephritis. Herein, we show that a monoclonal Id(LN)F(1)-expressing antibody, 540, significantly (P< or = 0.01) stimulated Id(LN)F(1)-reactive T-cell clones B6 and D2 to proliferate, while other Id(LN)F+1 antibodies did not. Further, injection of 540-producing hybridoma cells into nonautoimmune (SWRxBalb/c)F(1) mice resulted in the deposition of Id(LN)F(+)(1) Ig in the kidneys, in a pattern indicative of early nephritis. To identify the pathogenetic Id(LN)F(1) epitope(s) at the molecular level, we compared the deduced amino acid sequences of the heavy and light chain variable regions of pathogenetic and non-pathogenetic Id(LN)F(1)-expressing Igs 540, 317, and 533. Two overlapping peptides derived from the V(H) sequence of 540 (aa 54-66 and 62-73), which both contain the triple basic amino acid motif K(X)K(X)K, stimulated SNF(1) T cells and T-cell clones B6 and D2. These results further support the involvement of a subset of Id(LN)F(1)-expressing Ig in SNF(1) nephritis.