Painful diffuse osteosclerosis after intravenous drug abuse.

PURPOSE: We identify a new syndrome of acquired painful diffuse osteosclerosis associated with past intravenous drug abuse in two adults. METHODS: A 28-year-old white woman and a 38-year-old black man with a history of non-A, non-B chronic active hepatitis were referred to us for increasing bone pain that was especially severe in their lower extremities. They were studied at our clinical research center. RESULTS: Skeletal radiographs documented progressive generalized osteosclerosis. Increased bone mass was confirmed by dual-energy radiography, and bone scintigraphy showed diffusely increased radionuclide accumulation. Serum biochemical studies revealed elevated alkaline phosphatase activity and osteocalcin levels, mild to moderately increased 1,25-dihydroxyvitamin D concentrations, and normal parathyroid hormone levels. In urine, hydroxyproline excretion was elevated, whereas calcium levels were reduced. Iliac crest histomorphometry showed increased rates of bone formation. Hematology, renal function, serum protein electrophoresis, and screening for fluorosis as well as vitamin A and heavy metal poisoning were all normal. Family histories were negative. Both patients were seropositive for antibody against hepatitis C virus as well as against Epstein-Barr virus (antiviral capsid antigen IgG but not IgM). Each subject was seronegative for cytomegalovirus, human immunodeficiency virus (HIV) 1 and 2, and human T-cell lymphotropic virus (HTLV) 1 and 2. Assay for reverse transcriptase in lymphocyte co-culture fluid and polymerase chain reaction studies using HIV-1 primers on peripheral monocyte DNA were negative. Treatment with synthetic salmon calcitonin in both individuals rapidly led to decreased bone pain and to a decline in biochemical parameters of accelerated bone turnover. CONCLUSION: Painful diffuse osteosclerosis can follow intravenous drug abuse and is possibly caused by parenteral transmission of a virus that in some way stimulates bone formation.

The impact of ganciclovir-resistant cytomegalovirus infection after lung transplantation.

BACKGROUND: Cytomegalovirus (CMV) resistance to ganciclovir has become increasingly common in acquired immunodeficiency syndrome patients but has only rarely been reported in recipients of solid organ transplants. METHODS: A retrospective study of ganciclovir susceptibility testing of CMV isolates recovered from lung transplant recipients was performed. Patients with CMV isolates having partial (1 or =3 microg/ml) to ganciclovir determined by plaque reduction assay were included in a case-control study to identify risk factors for ganciclovir resistance. RESULTS: Between 2/91 and 5/98, 18 patients (5.2% of patients transplanted) were found to have CMV infections with some degree of ganciclovir resistance (4 partially, 14 fully resistant). More positive viral blood cultures (3.2+/-2.5 vs. 1.6+/-1.4 CMV positive cultures, P=0.02) and more episodes of CMV pneumonitis (0.24+/-0.23 vs. 0.10+/-0.17 episodes/bronchoscopy, P=0.02) occurring before the detection of resistance were seen among resistant patients than controls. Ganciclovir-resistant patients received more antithymocyte globulin during induction (70+/-44 vs. 45+/-39 mg/kg, P=0.03) and received ganciclovir for a greater number of days (79+/-52 vs. 64+/-53 days, P=0.005) before the detection of resistance than controls. Ganciclovir-resistant patients had a shorter survival and an earlier onset of bronchiolitis obliterans syndrome compared with patients in the transplant database at Washington University. CONCLUSIONS: Ganciclovir-resistant CMV infection is a serious complication of solid organ transplantation associated with more episodes of viremia, more frequent disease, earlier onset of bronchiolitis obliterans and shorter survival. The use of antithymocyte globulin and prolonged exposure to ganciclovir are risk factors for the development of ganciclovir resistance.

Oral acyclovir reduces the incidence of recurrent herpes simplex keratitis in rabbits after penetrating keratoplasty.

To determine if acyclovir sodium prevents postoperative herpes simplex virus type 1 (HSV-1) recurrences, 21 rabbits harboring latent HSV-1 underwent uniocular autograft penetrating keratoplasty. All operated-on eyes were treated with topical and subconjunctival dexamethasone sodium phosphate. Ten of the 21 rabbits also received oral acyclovir (intravenous acyclovir was given at the time of surgery). Postoperatively, 9 (82%) of 11 operated-on eyes in rabbits not treated with acyclovir had positive HSV-1 ocular cultures. In acyclovir-treated rabbits, however, none of the 10 operated-on eyes had positive ocular cultures. In addition, 9 (82%) of 11 of the operated-on eyes had geographic ulcers develop in the non-acyclovir-treated rabbits, compared with 1 (10%) of 10 in the acyclovir-treated rabbits. Finally, stromal keratitis appeared in 5 (56%) of 9 of the operated-on eyes in non-acyclovir-treated rabbits and 1 (12%) of 8 of the operated-on eyes in acyclovir-treated rabbits. The results of this study indicate that acyclovir significantly lowered the incidence of HSV-1 ocular shedding, geographic ulceration, and stromal keratitis in a rabbit autograft penetrating keratoplasty model.

Diagnostic testing for HIV type 1 RNA in seronegative blood.

We studied the feasibility of routine diagnostic testing for HIV-1 RNA at a publicly funded testing site. HIV-1 RNA was determined with a commercial polymerase chain reaction assay in pooled seronegative blood samples submitted for HIV testing to a public health laboratory. Recovery of HIV-1 RNA from the samples was estimated as at least 8% of viral RNA that was found in freshly prepared plasma. We estimated that screening for HIV-1 RNA in serum pools would result in the identification of blood specimens from more than 95% of acutely infected patients. The frequency of HIV-1 RNA in seronegative blood samples was estimated to be between 19 and 601 per 10(6) submitted specimens. The ratio of HIV-1 RNA positive and seronegative samples to specimens with HIV-1 antibodies confirmed by Western blot was estimated to be between 0.2% and 6.6%. The reagent costs for identifying 1 HIV-infected blood sample were 10-fold higher with the commercially available HIV-1 RNA assay compared with the HIV antibody enzyme-linked immunosorbent assay. Diagnostic testing for HIV-1 RNA may be warranted in high-risk populations since acutely infected patients may benefit most from anti-retroviral therapy and are thought to contribute disproportionately to the HIV epidemic.

Strategy for efficient detection of respiratory viruses in pediatric clinical specimens.

Direct immunofluorescence (IF) with a polyclonal respiratory syncytial virus (RSV)-specific antibody preparation was used for antigen detection during the 1982-1983 RSV season (155 specimens) and gave an overall sensitivity of 94% with 87% specificity compared with viral culture. Indirect IF was used in the 1983-1984 season (265 specimens) and exhibited sensitivity of 96% with specificity of 79%. During these two seasons, 42 of 224 (18.8%) specimens that were IF-negative for RSV grew viruses other than RSV. In the winter of 1984-1985, we screened 297 specimens for RSV by IF and 80 (27%) were positive. Forty-four (20%) of the IF-negative specimens were culture-positive for RSV(2) or other viruses(44). We conclude that, in the interest of cost reduction and expeditious detection of respiratory viruses, once a properly equipped laboratory has become thoroughly familiar with IF techniques, pediatric respiratory specimens can be screened for RSV by IF and only the IF-negative specimens need be inoculated into cell cultures for isolation of virus during the winter respiratory season.

Increasing prevalence of resistance mutations in antiretroviral-naïve individuals with established HIV-1 infection from 1996-2001 in St. Louis.

BACKGROUND: Transmission of drug-resistant virus in HIV-1 infected individuals is well documented, particularly in patients with primary infection. Prevalence in chronically infected antiretroviral-naïve patients is reportedly low. Routine genotyping in this population is not recommended. PURPOSE: The purpose of this study was to evaluate resistance profiles in patients with established HIV infection in St. Louis. METHOD: We selected specimens from drug-naïve individuals (CD4 >300 cells/mL and VL >1000 copies/mL) with established HIV infection between 1996-2001. 62 of 75 specimens were available for genotyping. We excluded patients with evidence of acute HIV infection and long-term nonprogressors. RESULTS: The overall prevalence of resistance was 11% (7/62). From 1996 to 1998, a prevalence of 4% was observed (1/27 individuals). During the subsequent period from 1999 to 2001, the frequency increased to 17% (6/35 participants; p =.08; 95% CI 5-29%). CONCLUSION: The results suggest that the prevalence of primary resistance is increasing in our region to the point that it justifies genotypic testing in all individuals before the initiation of antiretroviral therapy. This has to be considered when designing antiretroviral clinical trials.

Penetrating keratoplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used.

The increased incidence of corneal graft failure in patients with herpes simplex virus (HSV) keratitis may be due in part to reactivation of latent HSV following surgical corneal trauma and postoperative corticosteroid therapy. To determine the onset, frequency, and nature of HSV recurrences following penetrating keratoplasty (PKP), 21 HSV type 1 (HSV-1) latently infected rabbits underwent unilateral autograft PKP. Opposite unoperated eyes served as HSV-1 latently infected controls. Corneal autografts were performed so that immunologic graft rejection would not be confused with recurrent HSV-1 stromal disease. After PKP, 11 of the 21 eyes were treated with dexamethasone. Ocular cultures and slit-lamp examinations were performed daily for the first postoperative 8 days and every other day thereafter for 82 days. Nine (82%) of the 11 dexamethasone-treated PKP eyes, 2 (20%) of the PKP eyes not treated with dexamethasone, and 3 (17%) of the 18 unoperated eyes had positive HSV-1 ocular cultures. Geographic ulcers appeared only in the PKP eyes treated with dexamethasone; 9 (82%) of the 11 PKP eyes treated with dexamethasone developed geographic ulcers. Between the 24th and 90th postoperative days, stromal keratitis appeared in 5 (56%) of the 9 PKP eyes treated with dexamethasone and in 2 (25%) of the 8 PKP eyes not treated with dexamethasone. Autograft PKP with postoperative corticosteroids significantly increased HSV-1 ocular shedding, epithelial ulceration, and stromal keratitis. This experimental model provides a useful tool to further investigate the development and treatment of HSV-1 epithelial and stromal recurrences after PKP.

Value of rapid diagnosis of respiratory syncytial virus infection on management of small infants.

Respiratory syncytial virus (RSV) is a common cause of infection in infancy and early childhood. A presumptive diagnosis of RSV infection can frequently be made on clinical grounds. Confirmation can be made by viral culture, which may take 3 to 7 days. Immunofluorescent assay (IFA) is a specific and sensitive test that can provide laboratory confirmation of RSV infection the same day. Rapid diagnosis of RSV infection may have implications regarding prevention of nosocomial spread of RSV, early initiation of anti-viral therapy, use of antibiotics, and duration of hospital stay. Data are presented regarding the use of RSV-IFA and its effect on patient management.

Laboratory diagnosis of HIV infection.

The implications of a diagnosis of HIV infection are far-reaching. Thus, accurate interpretation of laboratory tests becomes critical. The authors provide descriptions of the tests now available and discuss the utility of each.

Simplified method for typing herpes simplex virus by restriction endonuclease analysis.

We have developed a simplified method for unambiguously typing herpes simplex virus. The method depends on the production of cell-associated virus at 34 degrees C and subsequently, on the separation of cellular DNA and viral DNA by Dounce homogenization and the removal of nuclei by centrifugation. Viral nucleic acid was prepared from the cytoplasmic fraction and analyzed after restriction endonuclease cleavage. The method obviates the use of radioactive isotopes, and the viral DNA is effectively free of interfering cellular DNA.

Problems associated with the use of (E)-5-(2-bromovinyl)-2'-deoxyuridine for typing herpes simplex virus.

When 0.5 microgram of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) per ml was incorporated directly into cell culture medium inoculated with eight known positive specimens, one herpes simplex virus type 1 (HSV-1) isolate grew in the presence of BVdU and was misidentified. By plaque assay, the titers of 15 HSV-1 strains were reduced by more than 3 log10 by BVdU, and the titers of 16 HSV-2 strains were reduced by less than 2 log10. Titers of HSV-1 acyclovir-resistant strains were reduced by less than 1.5 log10, which was characteristic of HSV-2 strains. Thus, typing of HSV isolates in the presence of BVdU by plaque assay is reliable only if information regarding previous antiviral therapy is obtained.

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.

Evaluation of two immunofluorescence assays with monoclonal antibodies for typing of herpes simplex virus.

An indirect immunofluorescence assay and a direct immunofluorescence assay were evaluated for typing clinical isolates of herpes simplex virus (HSV). The indirect immunofluorescence assay (Electro-Nucleonics, Inc.) correctly identified 16 HSV type 2 (HSV-2) isolates, but failed to identify 4 of 14 HSV-1 isolates because of background fluorescence and instability of reagents. Forty-nine HSV-1 isolates were correctly typed by direct immunofluorescence assay (Kallestad Laboratories, Inc.), but 1 of 39 HSV-2 isolates did not react with the HSV-2 type-specific antibody conjugate.

Effect of influenza B virus on nutrient transport in cultured epithelial cells.

The possibility that influenza virus could induce changes in membrane permeability to nutrients ordinarily concentrated within the cell was examined. Madin-Darby canine kidney cells were infected with egg-grown influenza B virus at 37 degrees C and pH 7.4 (a condition in which influenza virus enters cells by endocytosis). Control cells were mock-infected with allantoic fluid from chick embryos. Transport of phosphate, 2-deoxyglucose, and alpha-aminoisobutyric acid was measured at various intervals, 0 to 10 hours after infection. Uptake of alpha-aminoisobutyric acid and phosphate by infected cells was inhibited at 2 hours as compared with controls, whereas at 6 to 10 hours, the uptake of all nutrients was higher in infected cells. Infected cells preloaded with phosphate or 2-deoxyglucose did not demonstrate increased release of these nutrients. Thus, the virally induced inhibition of uptake early in infection is not a consequence of loss of membrane integrity. Transport studies were also performed in cells with prebound virus exposed to pH 5.0 for 60 seconds at 37 degrees C and then incubated at pH 7.4, at 37 degrees C. Under these conditions, influenza A viruses are known to enter the cell membrane by fusing directly with it and to initiate cell to cell fusion as well. We demonstrated that influenza B virus also caused cell fusion under these conditions. In contradistinction to studies described above at pH 7.4, fused, infected cells demonstrated both marked release and diminished uptake of nutrients as compared with controls. We conclude that influenza B virus does have an effect on host cell membrane permeability; the type of effect seen is markedly influenced by factors known to determine mode of virus entry into the cell.

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Silencing of human immunodeficiency virus long terminal repeat expression by an adenovirus E1a mutant.

Gene expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR) is strongly stimulated by the viral tat gene. The HIV LTR is also activated by several physical and chemical agents and heterologous viral genes, including adenovirus E1a. As E1a has separable transcriptional activation and repression functions, we examined the negative regulatory effects of E1a on the expression of the HIV LTR by using a trans-dominant E1a mutant. Mutant hr5 strongly suppressed the basal activity of the LTR as well as trans-activation of the LTR by heterologous agents such as the cytomegalovirus immediate early gene or DNA-damaging agents such as mitomycin C and UV irradiation. In addition, hr5 also caused significant suppression of tat gene-mediated trans-activation. The suppression of HIV LTR expression by hr5 appears to be mediated, at least in part, by the repression of the HIV enhancer, as the activity of an enhancer test system composed of the human T-cell leukemia virus I LTR containing an HIV-1 enhancer substitution was severely repressed by hr5. Cotransfection of HIV-1 proviral DNA with hr5 DNA resulted in a significant reduction of HIV production.