Absence of herpesvirus DNA by polymerase chain reaction in ocular fluids obtained from immunocompetent patients.

OBJECTIVE: To assess the prevalence of herpesvirus DNA in ocular fluids obtained from healthy patients undergoing vitreoretinal surgery. BACKGROUND: Polymerase chain reaction (PCR) has been used to detect herpesvirus DNA in patients with acute retinal necrosis and cytomegalovirus retinitis. Little is known regarding the prevalence of detectable herpesvirus DNA in ocular fluids collected from healthy seropositive patients with no clinical evidence of viral retinitis. METHODS: Seventy-five intraocular specimens (35 aqueous and 40 vitreous samples) were collected from 75 patients undergoing scleral buckling or vitrectomy. Using a PCR-based assay, the authors tested each specimen for the presence of herpesvirus genome DNA with primers specific for cytomegalovirus, Epstein-Barr virus, herpes simplex virus types 1 and 2, and varicella zoster virus. Serologic testing for immunoglobulin G (IgG) and IgM levels corresponding to each of the herpesviruses was also performed. RESULTS: Of the 75 samples tested, none was found to harbor herpesvirus DNA. The assay did not give false-positive results in patients with active intraocular inflammation. The sensitivity of the assay was 0.08 infection-forming units for cytomegalovirus, 0.6 tissue culture infectious doses for herpes simplex virus, 0.5 infected-cell equivalents for Epstein-Barr virus, and 0.03 focus-forming units for varicella zoster virus. The percentage of patients with positive herpesvirus serology ranged from 86% to 100% and was consistent with rates observed in the general population. CONCLUSIONS: The prevalence of herpesvirus DNA detectable by PCR techniques in ocular fluids appears to be quite low despite the high proportion of patients who tested positive for herpesvirus antibodies. Therefore, a positive result obtained in a patient presenting with vitreoretinal inflammation should be regarded as significant.

Laboratory consolidation. Minimizing the angst and aggravation.

In response to decreasing payment structures, many labs have begun considering those programs that strike fear in the hearts of employees everywhere--consolidation. To avoid whispers of layoffs, salary cuts, and scheduling disasters that become grist for the rumor mill, try constant communication and a structured management.

Stability of herpes simplex virus DNA in cerebrospinal fluid specimens.

Polymerase chain reaction methods are becoming the standard for the diagnosis of herpes simplex virus (HSV) encephalitis. Little is known, however, about the stability of HSV DNA in cerebrospinal fluid (CSF) specimens. Our results demonstrate that HSV DNA is extremely stable in CSF specimens containing lymphocytes and monocytes. We observed little DNA degradation in specimens stored for 30 days at room temperature (20-23 degrees C), refrigerator temperature (2-8 degrees C), or freezer temperatures (-18 to -22 degrees C and -69 to -72 degrees C). Specimen storage conditions are therefore not so critical for HSV encephalitis detection as for other viral illnesses. It is therefore not necessary to perform a second lumbar puncture to obtain a fresh specimen for the detection of HSV DNA.

Specimen collection and storage for diagnostic molecular pathology investigation.

The success of the newest discipline in the diagnostic clinical pathology laboratory, molecular pathology, is dependent on proper collection and storage of both the original and processed (nucleic acid) specimen. This issue will grow in importance as test volumes increase in the diagnostic molecular pathology laboratory. This review is a distillation of a literature review by the Patient Preparation and Specimen Handling Committee of the College of American Pathologists. It describes specific collection, storage, and anticoagulant or preservative requirements based on the diagnostic molecular technique and/or specimen type used for analysis. This review serves as a guide for clinical laboratories interested in appropriate collection and storage of specimens to be used in nucleic acid-based analysis.

Inhibition of PCR by aqueous and vitreous fluids.

The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology. While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicor carryover, relatively little attention has been given to the causes of false-negative PCRs. This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results. As little as 0.5 microliter of vitreous fluid and 20 microliters of aqueous fluid can completely inhibit DNA amplification in a 100-microliters PCR mixture. This inhibition was not primer specific, nor was it due to chelation of Mg2+ ions or DNase activity in the ocular fluid. The inhibitor was completely resistant to boiling for 15 min. However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction. The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction. Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant. Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases.

Molecular infectious disease testing: the hype, the hope, and the hoopla.

To remain competitive in the next century, microbiologists and virologists must acquire molecular diagnostic skills or they will relinquish molecular infectious disease testing to labs with little experience in handling infectious agents.

Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus viral capsid antigens.

Five enzyme immunoassays for the detection of Epstein-Barr virus immunoglobulin M were evaluated versus indirect immunofluorescence assays with 128 serum specimens. The sensitivities and specificities, respectively, of these kits were as follows: Gull, 92 and 100%; Incstar, 53 and 100%; Ortho, 89 and 100%; Sigma, 78 and 86%; and BioWhittaker, 94 and 70%. Indeterminate results were produced by 2, 2, 0, 8, and 14 specimens with the Gull, Incstar, Ortho, Sigma, and BioWhittaker tests, respectively. The Gull and Sigma tests had the best day-to-day reproducibilities, and the Gull test was the easiest to use. No correlation between immunofluorescence titers and enzyme immunoassay values was observed.

Improved isolation of Chlamydia trachomatis from a low-prevalence population by using polyethylene glycol.

The effect of polyethylene glycol (PEG) on the isolation of Chlamydia trachomatis was evaluated in our laboratory. Initial range-finding experiments demonstrated that the number of chlamydial inclusion bodies increased with increasing PEG concentrations. However, PEG concentrations above 10.5% became progressively more toxic to the McCoy cell monolayers. When 50 frozen clinical Chlamydia isolates were inoculated onto McCoy cell cultures with and without 7% PEG, the PEG-treated cultures produced three- to fivefold more chlamydial inclusions than cultures without PEG. This enhancement was also observed when 1,144 fresh clinical specimens from a low-prevalence population were tested. With fresh clinical specimens, PEG-treated cultures produced two- to sixfold more inclusions than standard cultures. The addition of 7% PEG to the chlamydial overlay medium significantly increased the number of inclusions in each culture, improved the sensitivity of the culture, and decreased the probability of missing a weakly positive specimen.

Interferon induced within the central nervous system during infection is inconsequential as a mechanism responsible for murine resistance to street rabies virus.

SJL/J mice are resistant, whereas A/WySnJ mice are susceptible to intraperitoneally (i.p.) inoculated street rabies virus (SRV). In this report we determine whether interferon (IFN) induced within the central nervous system (CNS) of these mice during infection is associated with resistance. We show that the high concentration of type 1 interferon (IFN-alpha/beta) within the CNS of A/WySnJ mice is ineffective in inhibiting SRV replication in these tissues, and is unimportant in ameliorating disease. More importantly, the 100% survival of SRV-infected SJL/J mice following neutralization of IFN within the CNS with anti-IFN-alpha/beta suggests that protection of target cells by this minimal amount of IFN is not the mechanism responsible for the innate resistance of SJL/J mice to i.p. inoculated SRV.

Latex agglutination test for detection of antibodies to Toxoplasma gondii.

A resurgence of interest in Toxoplasma gondii has occurred because this coccidian parasite causes lethal infections in immunologically compromised hosts and is responsible for at least 3,000 congenitally infected infants in the United States annually. Thus, rapid, specific, and inexpensive serologic tests are required for routine screening of patients, especially pregnant women. We have developed a latex agglutination test for antibodies to T. gondii which utilizes covalently coupled T. gondii antigens. When compared with an indirect immunofluorescence assay, the latex test had a sensitivity of 94% and specificity of 100%. Compared with an enzyme-linked immunosorbent assay, the latex test had 86% sensitivity and 100% specificity. When testing samples which exhibited nonspecific polar staining by the immunofluorescence assay, the enzyme-linked immunosorbent assay had a 50% false-positive rate, whereas the latex agglutination test yielded no false-positive results. Thus, the latex agglutination test provided an efficacious method for routine serological screening for antibodies to T. gondii.

Rat leukocyte interferons: production of an acid-labile interferon after induction with Newcastle disease virus.

Rat leukocytes produce three interferon species after infection with Newcastle disease virus. Two of the three interferon species (22-24,000 and 30,000 daltons) were acid and heat stable, protected rat and mouse cells from virus-mediated cytopathic effect (CPE), and were neutralized by antisera to mouse L-cell (alpha/beta) interferon. The third, 30,000-dalton interferon species was heat and acid labile; protected rat, mouse, human, and bovine cells from virus-mediated CPE; and was neutralized by antisera to mouse L-cell interferon and human leukocyte interferon. Thus, the rat leukocyte interferon system is similar to the human system in the kinetics of interferon production, the production of multiple interferons, heterologous antiviral activity, molecular weight, and in the production of an acid-labile species. The rat leukocyte interferon system therefore represents an important model for examining the normal production and action of acid-labile interferons and their possible role in the regulation of leukocyte function and cellular differentiation.

Analysis of molecularly cloned DNA reveals minor differences among three virus strains of Aleutian disease of mink parvovirus. Brief report.

Molecular clones representing a 1.55 kbp genomic segment from three strains of Aleutian disease parvovirus (ADV) were studied. All three clones directed synthesis of viral structural antigens. In addition, 19 of 23 restriction sites were shared among viruses.

Mink parvoviruses and interferons: in vitro studies.

Although interferons can inhibit the replication of a number of viruses, little is known about their ability to inhibit parvovirus replication. Therefore, in vitro experiments were done to determine if Aleutian disease virus and mink enteritis virus, two autonomously replicating mink parvoviruses, induced interferon, were sensitive to the effects of interferon, or inhibited the production of interferon. The results indicated that these parvoviruses neither induced nor were sensitive to the effects of interferon. Furthermore, preexisting parvovirus infections did not inhibit poly(I).poly(C)-induced interferon production. This independence from the interferon system may, therefore, be a general property of the autonomously replicating parvoviruses.

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.

Production and characterization of poly(I):poly(C)-induced rat interferons in vitro.

The synthetic polyribonucleotide polyinosinate:polycytidylate [poly(I):poly(C)] was used to induce interferon (IFN) production in a rat leiomyosarcoma cell line. Three separate IFN species were distinguished by antiviral activity on homologous and heterologous cells, interaction with Blue Sepharose CL-6B chromatography matrix, and antibody neutralization studies. Two IFN species, one with a weakly ionic and the other with a hydrophobic interaction with Blue Sepharose, exhibited heterologous antiviral activity, and were acid- and heat-labile. Both had a molecular weight of 30,000 daltons by HPLC-based molecular exclusion chromatography. The weakly ionic species was neutralized only by antiserum to L-cell IFN, whereas the hydrophobic species was neutralized by antiserum to L-cell IFN and antiserum to human leukocyte IFN. The remaining IFN species, with a weakly ionic interaction with Blue Sepharose, was acid- and heat-stable, exhibited no heterologous cell antiviral activity, and was not neutralized by any antisera to IFN. This species had a molecular weight of 39,000 daltons by HPLC-based molecular exclusion chromatography and 34,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These rat fibroblast IFNs possess greater heterogeneity than that reported or observed for murine or human fibroblast IFNs. These IFNs could thus be classified as follows: one species of rat IFN-beta and two different species of unique acid-labile rat IFNs.

Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies.

Previously it has been reported that strains of Rickettsia rickettsii that differ greatly in their ability to cause disease in guinea pigs are similar by serological and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. In this study, we used monoclonal antibodies to the virulent R and the relatively avirulent HLP strains to investigate strain differences which might account for the disparate behavior of the strains in guinea pigs. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the R and HLP strains were nearly identical for polypeptides with apparent molecular weights greater than 32 kilodaltons (kDa). All of the monoclonal antibodies to a lipopolysaccharide-like antigen reacted equally well with antigen from both strains by immunoblotting. None of the antibodies to the lipopolysaccharide-like antigen protected mice against challenge with viable rickettsiae. Some antibodies reacted with both 120- and 155-kDa polypeptides of both strains in radioimmune precipitation and immunoblotting tests, and other antibodies reacted only with the homologous strain. The monoclonal antibodies cross-reacted with the heterologous strain in the enzyme-linked immunosorbent assay essentially either completely or not at all. The ability of the monoclonal antibodies to the 120- and 155-kDa polypeptides to protect mice against the two strains was correlated with the ability of the antibodies to react with the antigens in the enzyme-linked immunosorbent assay and radioimmune precipitation or immunoblotting tests. These results demonstrate that R and HLP antigens which appear identical in molecular weight differ in their compositions of antigenic determinants.