Lymphogranuloma venereum and acute ulcerative proctitis.

The lymphogranuloma venereum (LGV) L2 serotype of Chlamydia trachomatis has been isolated from the rectums of three homosexual men with acute, primary ulcerative proctitis that responded to appropriate anti-chlamydial therapy. LGV is still present in the urban United States and must be considered in cases of acute ulcerative proctitis.

Antiviral activity and ocular kinetics of antisense oligonucleotides designed to inhibit CMV replication.

PURPOSE: To compare the antiviral activity and ocular distribution of first- and second-generation antisense oligonucleotides intended for the treatment of cytomegalovirus (CMV) retinitis. METHODS: The antiviral activity of ISIS 13312 and ISIS 2922 (Isis Pharmaceuticals, Inc., Carlsbad, CA) against 10 clinical CMV isolates was compared with a plaque-reduction assay. The ocular pharmacokinetics were compared after intravitreal injection in rabbits (36-90 microg) and monkeys (125-500 microg). Vitreous and/or retina were collected after single and multiple injections to characterize ocular distribution, clearance, and accumulation. Oligonucleotide concentrations were measured by capillary gel electrophoresis and immunohistochemical techniques. RESULTS: ISIS 13312 and ISIS 2922 demonstrated comparable antiviral activity that was consistent among the 10 clinical isolates examined (50% inhibitory concentration [IC(50)], <1 microM). Activity was independent of the resistance of CMV isolates to DNA polymerase inhibitors. After intravitreal injection, the kinetics of ISIS 2922 and ISIS 13312 were characterized by clearance from vitreous and distribution to the retina; however, ISIS 2922 was cleared more quickly from the retina than ISIS 13312. The half-life of ISIS 13312 in the monkey retina was approximately 2 months. Retinal concentrations of ISIS 13312 were dose dependent, with approximately a twofold increase in concentration after once-monthly doses compared with single-dose concentrations. Immunohistochemical analysis indicated that both oligonucleotides were efficiently distributed to numerous ocular tissues, including retina, ciliary body, and optic nerve. CONCLUSIONS: ISIS 13312 possesses antiviral activity and pharmacokinetic properties that favor its use as a therapeutic agent in treatment of CMV retinitis. The half-life of ISIS 13312 in retina is longer than that of ISIS 2922, potentially allowing for less frequent administration.

Variations in the cytomegalovirus DNA polymerase and phosphotransferase genes in relation to foscarnet and ganciclovir sensitivity.

BACKGROUND: Identification of human cytomegalovirus (CMV) genome variation is important for understanding mutations associated with drug resistance. OBJECTIVES: To investigate the CMV resistance to foscarnet (PFA) and ganciclovir (GCV) in patients treated with antiviral drugs and to identify the DNA polymerase (UL54) and phosphotransferase (UL97) gene mutations inducing resistance. STUDY DESIGN: Antiviral susceptibility of CMV strains/isolates for PFA and GCV was compared by plaque reduction assay and in situ ELISA. UL54 and UL97 gene mutations were identified by sequencing. Growth phenotype of two CMV recombinants with mutations in UL54 was studied. RESULTS: Six of seven GCV resistant strains had alterations within the UL97. Five of them also had alterations in the UL54 (F412C, L802M or K513E), previously shown to induce GCV resistance. Seven isolates had no or reduced susceptibility to PFA, which had alterations in the UL54 (D588N, E756K, V781I or L802M). By in vitro mutagenesis, it was shown that a mutation at codon D588N of UL54 conferred 9-fold reduced susceptibility to PFA, while a mutation at codon V781I induced 4-fold reduced susceptibility to PFA and GCV. Both recombinants showed the same kinetics of protein expression (IE, E, and L antigen) and virus yields as the CMV Towne strain. CONCLUSIONS: The recombinants containing alterations within the UL54 (D588N and V781I) showed a reduced susceptibility to antiviral drugs but no change in the replication rate compared to the CMV Towne.

Direct inactivation of herpes simplex virus type-2 by rat epidermal protein.

Proteins were extracted from corneocytes of skin of 2-day-old rats and fractionated by gel filtration and cation exchange column chromatography. The different protein fractions were tested for direct inactivation of herpes simplex virus infectivity as determined by reduction of plaque formation. The most effective protein fractions against herpes simplex virus were a neutral pH buffer soluble and mol. wts. ranging from 20 K to 30 K. Amino acid composition of the proteins were virtually identical to epidermal histidine-rich proteins. The activity was significantly (P less than 0.001) stronger against type-2 than type-1. The activity was most stable at pH 7.2 and the rate of inhibition increased in a time-dependent manner up to 4 h. The 50% effective dose was estimated as 1.1 micrograms protein/ml.

Transfusion-related cytomegalovirus infection following noncardiac surgery.

Clinically evident cytomegalovirus (CMV) infections developed in five patients after noncardiac surgery. Each of them had received several blood transfusions and had evidence of primary CMV infection. These five cases were identified in a single eight-month period, suggesting that this syndrome must be occurring with greater frequency than is commonly appreciated. Three of five patients had undergone splenectomy, and review of the literature for documented cases of posttransfusion CMV mononucleosis following non-cardiac-bypass surgery disclosed an additional nine patients, five of whom had had splenectomies. This striking association suggests the possibility that the spleen plays a role in controlling the incidence or clinical manifestations of posttransfusion CMV infections. Alternatively, this association may only reflect the frequent requirement for many blood transfusions in this type of surgery.

A deletion mutation in region V of the cytomegalovirus DNA polymerase sequence confers multidrug resistance.

A patient with AIDS and cytomegalovirus (CMV) retinitis received ganciclovir and foscarnet for 20 and 5 months, respectively, with evidence of periodic disease progression. After this therapy, a CMV isolate from the patient was resistant to ganciclovir, foscarnet, and cidofovir. Sequence analysis showed a known ganciclovir resistance mutation in the viral UL97 phosphotransferase (L595F) and a new mutation in conserved region V of the DNA polymerase gene (pol) sequence (codons 981-982 deleted). The pol mutation was transferred to a laboratory CMV strain (Towne) by homologous recombination and selection with either ganciclovir or foscarnet. Recombinant viruses containing this deletion showed a 6-8-fold increased ganciclovir resistance and a 3-5-fold increased resistance to both foscarnet and cidofovir, compared with the wild-type CMV. A single mutation in region V of CMV pol can, therefore, confer multiple drug resistance in a clinical isolate.

Anticomplement immunofluorescence test that uses isolated fibroblast nuclei for detection of antibodies to human cytomegalovirus.

Cytomegalovirus antibodies were measured in human sera by a nuclear anticomplement immunofluorescence test that used as antigen the isolated nucleic of virus-infected fibroblasts cells lysed in distilled water. The method exhibited less nonspecific fluorescence than either a conventional whole-cell anticomplement immunofluorescence test or an indirect fluorescent antibody test applied to the same isolated nuclear substrate. The assay detected 97.5% of 40 antibody-positive sera, compared with 92.5 and 90% detection rates by indirect hemagglutination and complement fixation, respectively. In addition, antibody titers obtained by this technique were significantly higher than those obtained by either indirect hemagglutination (P < 0.02) or complement fixation with a glycine-extract antigen (P < 0.001).

Evaluation of the virus permeability of a new condom for women.

The authors tested a polyurethane women's condom for permeability to the human immunodeficiency virus (HIV) and cytomegalovirus (CMV) using an artificial intercourse model. They did not detect viral leakage in three trials for each virus. Use of this device, which can be controlled by the woman, may reduce HIV and CMV infection.

PIP: In San Francisco, California, researchers used an artificial intercourse model to test a polyurethane woman's condom (WPC-33 under development by the Wisconsin Pharmaceutical Company) for permeability to HIV and cytomegalovirus (CMV). The disposable device is loose-fitting and has flexible rings at both ends. The researchers placed a virus suspension inside a condom and then placed inside it a second condom containing tissue culture medium to test for permeability. A 35-ml plastic syringe case served as an artificial penis. It was inserted into the inner condom, which was inside an artificial foam vagina, and then plunged 50 times to simulate trauma associated with sexual intercourse. Aliquots of the fluids in the outer and inner condoms were grown in culture. CMV was grown in human embryonic lung fibroblasts for four weeks. Virus-positive cultures were identified by their cytopathogenic effect. HIV was grown for eight days in normal human lymphocytes and assayed with an antigen capture enzyme-linked immunosorbent assay (ELISA). In three trials using CMV, all inner condom fluids were positive; all outer condom fluids were negative. The detection limit of the assay was 0.2 mcl of inoculum. The same results were found in three trials with HIV. The detection limit of the ELISA for HIV was 0.67 mcl of inoculum. Thus, a female condom may present an alternative means of controlling sexually transmitted diseases and conception. Previous studies have suggested that the female condom may leak less than a male condom. However, this device has not yet proven effective in vivo and it was not tested for efficacy as a contraceptive. Additional studies will be required to prove the utility of this barrier in vivo.

Frequency of UL97 phosphotransferase mutations related to ganciclovir resistance in clinical cytomegalovirus isolates.

Cytomegalovirus (CMV) isolates from subjects who received ganciclovir therapy were tested for susceptibility to ganciclovir by a plaque reduction assay. Results were correlated with restriction enzyme and sequence analysis of the CMV UL97 phosphotransferase gene. Of the 30 isolates, 20 had one or more mutations in UL97 affecting amino acid encoding at codons 460, 520, or 591-596. All 20 were resistant to ganciclovir, with an IC50 of > 6.0 microM (range, 6.7-50.0). The remaining 10 isolates had no mutations at these loci; 8 were susceptible to ganciclovir while the other 2 were borderline resistant (IC50s, 6.6 and 7.2 microM). None of 40 control CMV isolates from untreated subjects contained any amino acid changes at these loci. The three most common mutations at codons 460, 594, and 595 were detected by restriction digest analysis in 16 (80%) of 20 isolates and in 16 (73%) of 22 isolates with ganciclovir IC50s > 6.0 microM. These results indicate that the majority of ganciclovir-resistant clinical isolates contain diagnostically useful mutations in UL97.

Failure of high-dose oral acyclovir to suppress CMV viruria or induce ganciclovir-resistant CMV in HIV antibody positive patients.

Ninety-three symptomatic HIV antibody positive patients were randomized to receive zidovudine (ZDV) 600 mg/day and acyclovir (ACV) 4,800 mg orally per day versus ZDV 600 mg/day plus placebo. Urine was obtained at 3-month intervals and cultured for cytomegalovirus (CMV) in diploid fibroblast cells. The percent of urine specimens positive for CMV was 7.1% in the ZDV group and 5.8% in the ZDV plus ACV group (p = 0.55); 27% of patients had at least one urine culture positive for CMV while taking ZDV, versus 20% of patients taking the combination of ZDV plus ACV (p = 0.52). We conclude that ACV at a dosage of 4,800 mg/day does not suppress CMV excretion in urine of symptomatic HIV antibody positive patients taking concurrent ZDV. Use of ACV did not appear to induce resistance of CMV to ganciclovir since the ID50 of isolates from the two treatment groups did not differ.

Cytomegalovirus infections in homosexual men. An epidemiological study.

Levels of cytomegalovirus antibody (IgG and IgM) were measured and urine viral cultures were done in 237 homosexual men over a mean period of 14.1 months. The initial prevalence of cytomegalovirus IgG antibody was 86.9%. By the 9th month of follow-up, 71% of serosusceptible men had become infected with cytomegalovirus. During the study period cytomegaloviruria was noted in 32% of seropositive men. Cytomegalovirus IgM antibody was intermittently present in the serum of 95% of IgG-seropositive men, suggesting that frequent reactivation of latent infection or reexposure to exogenous virus had occurred. Of seven sexual practices investigated, only passive anal-genital intercourse correlated with the acquisition of cytomegalovirus infection (p = 0.008).

Prevalence of cytomegalovirus infection in homosexual men.

Cytomegalovirus (CMV) was cultured from the urine of 14 of 190 homosexual but none of 101 heterosexual men attending a venereal disease clinic (P less than 0.005). Viruria was confined to men less than 30 years of age and was present in 14% of this group. Antibody to CMV was measured in the sera of 139 homosexual and 70 heterosexual men attending the same clinic and in 103 male volunteer blood donors. Titers were found in 94% of homosexual patients but in only 54% of heterosexual patients (P less than 0.005) and 43% of male volunteer blood donors (P less than 0.005). The data suggest that sexual transmission is an important mode of spread of CMV among adults and the homosexual men are at greater risk for CMV infections than are heterosexual men. Homosexual men might considered candidates for the evaluation of the efficacy of CMV vaccines in preventing horizontal transmission of infection.

Simultaneous detection of antibodies to cytomegalovirus and herpes simplex virus by using flow cytometry and a microsphere-based fluorescence immunoassay.

A sensitive assay for the simultaneous detection of anti-cytomegalovirus and anti-herpes simplex virus antibodies was developed. Two different sizes of polystyrene microspheres were coated with purified viral antigens. Human antiviral antibodies were detected with a biotin-streptavidin amplification procedure with phycoerythrin as the fluorescent label. Microsphere-associated fluorescence was quantitated with a flow cytometer. Sixteen percent of samples initially scored as seronegative for cytomegalovirus and 35% of samples initially scored as seronegative for herpes simplex virus by conventional assays were clearly found positive by the microsphere technique. This flow cytometric assay can simultaneously detect several specific antibodies at levels which are below the sensitivity of standard assays. The dynamic range of this assay is at least sixfold greater than that of enzyme immunoassays. This technique is amenable to numerous serologic assays and could greatly expand the clinical laboratory applications of flow cytometry.

High-dose acyclovir compared with short-course preemptive ganciclovir therapy to prevent cytomegalovirus disease in liver transplant recipients. A randomized trial.

OBJECTIVE: To assess the efficacy of high-dose oral acyclovir therapy compared with preemptive, short-course ganciclovir therapy (administered only if cytomegalovirus [CMV] shedding occurred) to prevent CMV disease in liver transplant recipients. DESIGN: A randomized controlled trial. SETTING: Liver transplant center at a university-affiliated Veterans Affairs Medical Center. PATIENTS: 47 consecutive patients having liver transplantation. INTERVENTION: Patients were stratified by their CMV antibody status and the CMV antibody status of the donor and were randomly assigned to one of two treatment groups. Surveillance cultures for CMV (buffy coat and urine) were done every 2 to 4 weeks for 24 weeks in all patients. One group received high-dose oral acyclovir (800 mg four times daily). The experimental group received no acyclovir, but if surveillance cultures were positive, ganciclovir (5 mg/kg intravenously twice daily) was administered for 7 days. MEASUREMENTS: Cytomegalovirus shedding and CMV disease were measured in the two groups. RESULTS: Cytomegalovirus shedding before the onset of CMV disease occurred in 25% (6 of 24) of patients in the acyclovir group compared with 22% (5 of 23) in the experimental group. Cytomegalovirus disease developed in 29% (7 of 24) of the acyclovir group and in 4% (1 of 23) of the experimental group (P < 0.05). No hematologic toxicity occurred with ganciclovir. CONCLUSION: Oral acyclovir is ineffective prophylaxis against CMV in liver transplant recipients. Preemptive, short-course ganciclovir therapy in patients with CMV shedding was well tolerated and provided effective prophylaxis against subsequent CMV disease; this protocol targets the patients at risk for CMV disease and minimizes toxicity and expense.

Loss of rubella antibody from immune globulin treated with kaolin.

Sera and immune globulin (IG) preparations are customarily treated with kaolin before titration of their rubella hemagglutination-inhibiting (HI) antibody in order to rid them of nonspecific inhibitors of hemagglutination. The treatment was shown in this investigation to have no adverse effect on the antibody level of the sera but was found to remove considerable amounts of gamma-globulin from IG preparations. Evidence of this removal was obtained by serological tests, by spectrophotometric determination of protein concentration and by disc electrophoresis. In contrast to kaolin, heparin-manganese chloride (MnCl(2)) treatment of IG preparations had essentially no effect on the level of antibody globulin by all the criteria used. Heparin-MnCl(2)-treated IG lots were in these respects similar, if not identical, to their untreated counterparts. Since nonspecific inhibitors associated with the beta-lipoprotein fraction of serum are removed by the method employed to fractionate the IG samples, it seems unnecessary to treat the latter in any way for the HI test. No difficulty was encountered in this investigation with several untreated IG lots.

Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease.

In a human immunodeficiency virus-infected subject, cytomegalovirus (CMV) isolated 9 months after the patient began oral ganciclovir prophylaxis was resistant to ganciclovir and cidofovir and contained mutations in both UL97 and Pol coding regions. At 1 year, retinitis developed, which progressed despite intravenous ganciclovir followed by foscarnet and then cidofovir. A subsequent buffy coat virus isolate was resistant to all three drugs and contained new mutations in UL97 and Pol. By individually transferring the observed mutations to laboratory strain AD169, it was shown that a mutation at codon 603 of UL97 conferred resistance to ganciclovir, a mutation at codon 412 of Pol conferred resistance to both ganciclovir and cidofovir, and a mutation at codon 802 of Pol conferred resistance to ganciclovir and foscarnet. This case illustrates the development of multidrug resistance during prolonged exposure to antiviral therapy for CMV and cross-resistance arising from point mutations in the CMV Pol gene.

Mutation in region III of the DNA polymerase gene conferring foscarnet resistance in cytomegalovirus isolates from 3 subjects receiving prolonged antiviral therapy.

Three human immunodeficiency virus-infected subjects with progressive cytomegalovirus (CMV) retinitis despite prolonged antiviral therapy had buffy coat CMV isolates that were resistant to both ganciclovir and foscarnet. Genetic analysis of the resistant isolates showed that each contained a well-known ganciclovir resistance mutation in the viral UL97 phosphotransferase sequence, as well as a mutation (Ala to Val at codon 809, V809) in conserved region III of the DNA polymerase (Pol) sequence. A segment of the Pol sequence from one of the clinical isolates was transferred to CMV laboratory strain AD169 by homologous recombination. The recombinant virus containing V809 showed 6.3-fold increased foscarnet resistance and 2.6-fold increased ganciclovir resistance. Occurrence of the V809 mutation in 3 unrelated cases suggests that it is a clinically significant viral genetic marker for foscarnet resistance and decreased susceptibility to ganciclovir.

Prevalence of resistance in patients receiving ganciclovir for serious cytomegalovirus infection.

Seventy-two AIDS patients treated with ganciclovir for cytomegalovirus (CMV) disease were prospectively monitored for the development of drug-resistant virus. No resistant strains were found in 31 patients before therapy or among seven culture-positive patients treated for less than or equal to 3 months. Of 13 culture-positive patients treated for greater than or equal to 3 months, 5 excreted virus resistant (ED50, greater than 12 microM, or ED90, greater than 30 microM) to ganciclovir. Thus, 38% of patients and receiving ganciclovir for greater than 3 months and excreting virus or, overall, 7.6% of the patients were excreting CMV resistant to the drug.

Quantification of cytomegalovirus DNA in peripheral blood leukocytes by a branched-DNA signal amplification assay.

Quantification of cytomegalovirus (CMV) DNA in blood may aid in the identification of patients at highest risk for developing CMV disease, the evaluation of new therapeutics, and the prompt recognition of drug-resistant CMV strains. A branched-DNA (bDNA) assay was developed for the reliable quantification of CMV DNA in peripheral blood leukocytes. The bDNA assay allowed for the highly specific and reproducible quantification of CMV DNA in clinical specimens. Furthermore, the bDNA assay was at least as sensitive as culture techniques and displayed a nearly 3 log10 dynamic range in quantification. Changes in CMV DNA levels measured by the bDNA assay in a human immunodeficiency virus-positive patient undergoing therapy were consistent with CMV culture, antigen, and genotype results and correlated with disease progression and resistance markers. The bDNA assay for the quantification of CMV DNA may provide a useful tool that can be used to aid physicians in monitoring disease progression, evaluating therapeutic regimens, and recognizing viral resistance and drug failure.

Outbreak of Burkitt's-like lymphoma in homosexual men.

Four cases of Burkitt's-like lymphoma (undifferentiated, monoclonal, B-cell tumours) in homosexual men were seen in a 9-month period in San Francisco. One tumour contained both Epstein-Barr-virus nuclear antigen (EBNA) and cytomegalovirus (CMV) antigen. Another tumour contained EBNA, and a third contained no viral antigen, but EBNA and CMV antigens were detected in the overlying epithelium. This outbreak widens the array of neoplasms affecting immunosuppressed homosexual men and provides further evidence of an oncogenic role for EBV and CMV.