Comparison of hepatitis C viral loads in patients with or without human immunodeficiency virus.

A better understanding of how human immunodeficiency virus (HIV) coinfection affects the course of hepatitis C virus (HCV) infection is required to select patients with HIV who would benefit from current HCV therapy. Between June 1996 and March 2000, HCV RNA levels were quantified for 1,279 patients at the Louisiana State University Health Sciences Center; 28 of these patients were coinfected with HIV. HCV loads were quantified by the Bayer branched-DNA assay with a lower limit of detection of 0.2 Meq/ml. We compared the median HCV RNA levels of for patients coinfected with HIV and HCV and patients infected only with HCV who were in the same age range (23 to 55 years). The median HCV load for the 28 patients coinfected with HCV and HIV (17.8 Meq/ml) was significantly greater (P < 0.05) than that for similarly aged patients infected only with HCV (6.1 Meq/ml). The HCV load did not correlate with age or sex for either group of patients. A significant (R = -0.4; P < 0.05) negative correlation was observed between HCV load and CD4 count in the coinfected group, for whom the CD4 counts at the time of HCV load analysis ranged from 6 to 1,773/mm(3). The increased HCV load in patients coinfected with HCV and HIV compared to that in patients infected only with HCV and the inverse relationship of the HCV load to the CD4 count indicate that immunosuppression results in decreased control of HCV replication. In addition, we report significantly higher HCV loads among coinfected African Americans than Caucasians.

Validation of the Roche COBAS Amplicor system for Chlamydia trachomatis.

OBJECTIVE: Validation of the Roche Amplicor polymerase chain reaction (PCR) using the Comprehensive Bio-Analytical System (COBAS) automated PCR analyzer in our laboratory. DESIGN: Endocervical swab specimens for both EIA and PCR were collected from a total of 193 women. EIA for chlamydia was performed using the MicroTrak Chlamydia Kit (Wampole Labs, Cranbury, NJ). PCR was performed using Roche Amplicor reagents on the COBAS instrument. SETTING: Louisiana State University Health Sciences Center at Shreveport, Shreveport LA. PATIENTS: All cervical swab specimens, (n = 193), collected from patients presenting either to the Women's Health or Primary Care Clinic (Obstetrics and Gynecology and Family Practice) were included in this study. RESULTS: Most of the specimens, 138/193 or 71.5%, tested negative by both techniques. Three of the 193 specimens, 1.5%, were inhibitory for PCR since the internal control was negative. Fifty-one specimens, 26.4%, tested positive by both techniques or by PCR alone. No specimens were positive by EIA only. Twenty-eight of the 51 were positive by both methods, (14.5% of the total tested; 54.9% agreement among the specimens testing positive). An additional 23 were positive by PCR alone, i.e., 11.9% total discrepant positive specimens; 45% discordant results among the specimens testing positive). Seventeen PCR-positive specimens divided among four separate runs were retested by PCR. Of these, 15 were repeat positive, giving the test a reproducibility of 88.2%. CONCLUSIONS: Our results concur with previously published comparison data for EIA and PCR testing. We conclude that the PCR should detect a significantly increased number of chlamydia infections among our LSUHSC-S population, but there are drawbacks to using this technique. Specimen preparation time for PCR is almost twice as long as EIA, and the Roche PCR assay is not licensed for ocular specimens as is our EIA procedure. In addition, since neither technique is accepted for testing for medicolegal purposes, we must continue the use of culture for cases of suspected sexual abuse.

Clinical laboratory evaluation of a PCR assay for the detection of HCMV.

OBJECTIVE: In limited laboratory space and consonant with limited opportunities for contamination, to implement and incorporate into our diagnostic virology laboratory, a polymerase chain reaction assay for human cytomegalovirus detection with maximum sensitivity. DESIGN: Polymerase chain reaction, adapted for use with the enzyme uracil-n-glycosylase to avoid the potential for false positive reactions due to amplicon carryover was developed, optimized using two primer pairs, and performed on 361 specimens, i.e., body fluids and tissues submitted to the viral laboratory for detection of human cytomegalovirus. Polymerase chain reaction results were compared to shell vial assay. SETTING: Louisiana State University Health Sciences Center, Shreveport LA. MAIN OUTCOME MEASUREMENTS: Using the shell vial assay as the reference, analytical sensitivity (lower limit of detection) as well as laboratory sensitivity and specificity of both primer pairs. RESULTS: The lower limit of detection of our polymerase chain reaction assay was determined to be one focus-forming unit. Using the shell vial assay as the reference test, the sensitivity and specificity of both primer pairs were 96.5% and 94.3%, respectively. Polymerase chain reaction detected human cytomegalovirus in 6% of our culture-negative specimens. CONCLUSION: Based upon this study, our recommendations include the following: 1) a housekeeping gene amplification control is required for diagnostic polymerase chain reaction; 2) a single primer pair can be utilized for clinical work without sacrificing too much sensitivity; and 3) the laboratory should maintain close contact with clinicians to discuss polymerase chain reaction interpretation.

An in vitro assessment of the antineoplastic potential of 2H-1,3-oxazine-2,6(3H)-dione (3-oxauracil), a novel pyrimidine.

The pyrimidine (uracil) analogue 3-oxauracil (OU) previously had been shown to completely inhibit the growth of E. coli B and decrease by 96% the replication of herpes simplex virus type 2 when present in the culture fluid at a concentration of 10(2) microM. Limited in vivo studies in mice demonstrated antiviral effects without significant toxicity when given i.p. daily for two weeks at a concentration of 3.23 mg/kg. However, the antineoplastic properties of OU were unknown. We assessed the ability of OU to inhibit the proliferation of various human tumor cell lines (3 pancreatic, 1 colon, 1 neuroendocrine, and 1 lung) in an in vitro radiometric (Bactec) system. In the pancreatic lines (RWP-2, MiaPaCa-2, and PANC-1), the colon line (HT-29), the neuroendocrine line (COLO 320DM), and the lung cancer cell line (SK-MES-1), OU at a concentration of 10(3) microM, produced a dramatic decrease in percent cell survival. When compared with cytotoxic drugs of choice for these tumor cells (gemcitabine, 5-fluorouracil, and adriamycin, respectively) a significantly higher concentration of OU was required usually to achieve comparable results with two exceptions. These were the HT-29 and the COLO 320DM cell lines. These results indicate OU has significant (p < 0.05) cytotoxic activity against pancreatic, colon, neuroendocrine, and nonsmall cell lung cancer lines, when compared to untreated control cultures. Additional in vivo testing of this potential antineoplastic agent is warranted.

Lack of effect of respiratory syncytial virus infection on theophylline disposition in children.

Conflicting reports raise a question about decreased plasma clearance (Clp) of theophylline in man during viral infections. Thus a dilemma exists concerning requisite dose adjustments. We examined this issue by retrospectively evaluating theophylline Clp in children infected with respiratory syncytial virus (RSV). Two pharmacokinetic approaches were applied to a one-compartment open model to fit theophylline concentrations during 83 hospitalizations of 76 children, 6 to 48 months of age, who received intravenous theophylline therapy and were tested for RSV infection. Iterative linear regression analyses of all theophylline data were used to estimate apparent volume of distribution, elimination rate constant, plasma half-life, and Clp in 39 of the hospitalizations. When insufficient data were available to distinguish apparent volume of distribution and elimination rate constant (n = 44), steady-state estimates of Clp were calculated. An age-matched and percentile body weight-matched cohort design presented RSV as the primary covariate. Theophylline Clp was similar in 29 matched RSV-infected and -uninfected pairs (1.32 +/- 0.14 and 1.25 +/- 0.05 ml/kg per minute, respectively), as were other pharmacokinetic values. Unexpectedly, a significant, inverse linear relationship was found for Clp and percentile body weight. Additionally, children born prematurely and hospitalized in the neonatal intensive care unit had significantly higher theophylline Clp; this did not affect findings regarding RSV infection. Theophylline Clp was not decreased in RSV-infected children. Current theophylline dosing recommendations for young children infected with RSV should not be altered, but careful monitoring of plasma theophylline levels should be continued.

Summertime respiratory syncytial virus infection: epidemiology and clinical manifestations.

Respiratory syncytial virus (RSV) is a common serious pathogen known to produce annual winter epidemics in young children. A 2-year study of children with significant respiratory disease during the summer revealed a 21% incidence of RSV infection. Respiratory secretions collected from ill children in the LSUMC outpatient clinics, from children seen by private physicians, and from children hospitalized with respiratory tract disease were assayed for RSV antigens. Approximately 39% of those surveyed in 1987 and 13% of those studied in 1988 were positive. As this prevalence was significant, we compared RSV-induced disease in 20 patients hospitalized in summer and 20 hospitalized in winter (1989). The patients were matched by age, weight, sex, and race. Comparisons included subjective severity of disease, presenting symptoms, physical findings, chest roentgenograms, treatment, and average length of hospital stay. No significant differences in disease severity and/or clinical presentation were found. Our findings show that RSV induces disease in the summertime more frequently than generally recognized, and severe disease requiring hospitalization is not infrequent. Physicians should consider RSV in children with serious respiratory disease throughout the year.

Respiratory syncytial virus infection among intubated adults in a university medical intensive care unit.

Respiratory syncytial virus is the major cause of lower respiratory tract infection in children. Adults who are immunocompromised, aged, institutionalized, and/or have underlying medical diseases may be at risk for severe RSV infection. Intubated adults in an MICU were evaluated for evidence of RSV infection. Respiratory secretions were analyzed by cell culture and RSV EIA. Serologic testing was obtained. Respiratory secretions from MICU personnel with acute respiratory symptoms and patients admitted for pneumonia, asthma, or COPD also were screened. Five of 11 intubated patients had evidence of RSV infection. One of seven MICU employees and four of 48 ward patients had RSV-positive respiratory secretions. During community outbreaks of RSV infection, adults admitted to an MICU already may be infected with RSV; those admitted for other reasons are at risk for nosocomial infection. Patients occupying other hospital units and personnel may be instrumental in the nosocomial dissemination of RSV.

A structural model for the genome of echovirus 22.

We have proposed previously that the structural model for the echovirus 22 genome is a single-stranded RNA molecule that has folded back upon itself to form a stable "hairpin" at the 5'-terminus. The vRNA of echovirus 22 has been characterized further by digestion with selective ribonucleases, electrophoresis in composite gels, hydrodynamic studies in density gradients of Cs2SO4 and sucrose, thermal denaturation and 3'-terminal ribonucleotide analysis. Based on these observations, the genome of echovirus 22 is a single-stranded RNA molecule having a region of secondary structure located at the 5'-terminus that may be characterized as a snapback hairpin with hydrogen-bonded base-pairing. In addition, a VPg-like protein is attached (presumably to the 5'-end of the RNA) and the 3'-terminus contains a polyadenylic acid tract [poly (A)].

Evidence for secondary structure within the virion RNA of echovirus 22.

Phenol-extracted echovirus 22 virion RNA is infectious, but unlike poliovirus virion RNA, it resists digestion with pancreatic RNase and nuclease P-1, a 3' exonuclease selective for single-stranded RNA. These data indicate the presence of an enzyme-resistant portion somewhere in the RNA molecule and suggest that it is a double-stranded or base-paired region distant from the unblocked 3' terminus. Equilibrium density gradient centrifugation of native echovirus 22 virion RNA results in a single peak with a density of 1.63 g/cm3. When sheared before centrifugation, the molecule is resolved into two RNA species: one with an approximate density of 1.70 to 1.71 g/cm3, as is observed also for single-stranded poliovirus virion RNA, and the other with a density of 1.58 to 1.59 g/cm3. Data obtained from rate zonal centrifugation may be used to calculate an approximate sedimentation coefficient corrected to water at 20 degrees C of 34 and a molecular weight of 2.4 X 10(6) for the virion RNA. We propose a model for echovirus 22 RNA composed of a linear RNA molecule with a 5' hairpin.

Coxsackievirus B4: in vitro genetic markers and cardiovirulence. Brief report.

The relationship of cardiovirulence in mice (HAM/ICR, Swiss ND-4) of four coxsackievirus B4 strains to previously described in vitro markers (t, d, DS, DEAE-D, G, PS) has been determined. A relationship between plaque size and acardiovirulence was demonstrated in the HAM/ICR coxsackievirus B4 mouse system.

Evaluation of the effectiveness of 2H-1,3-oxazine-2,6(3H)-dione (oxauracil) in the treatment of herpes simplex virus (HSV)-induced acute encephalitis in mice.

Treatment of suckling mice with the pyrimidine analogue 2H-1,3-oxazine-2,6(3H)-dione (oxauracil) proved successful in reducing mortality associated with HSV-2-induced encephalitis. Oxauracil did not decrease mortality associated with HSV-1 infection; however, there was an increased survival time and a decrease in the amount of infectious virus recovered from the brains of HSV-1-infected animals.

Coxsackievirus B4: in vitro genetic markers and virulence.

The relationship of virulence to mice of the coxsackievirus B4 prototype JBV strain and selected mutants to several in vitro genetic markers has been examined. Markers studied include the t, d, DS, DEAE-D, and guanidine resistance (G), markers and a plaque size marker, here designated PS. The JVB strain was found to have the characteristics t-, d-, DS+, DEAE-D+, G-, PS+ and to be virulent when inoculated by the intraperitoneal route into suckling CD-1 mice (log titer/LD50 = 6.4/6.0). One naturally occurring mutant strain (characteristic = t-, d+, DS+, G-, PS+ was selected by terminal end point dilution and found to be virulent (log titer/LD50 = 7.0/6.0). Passage of the JVB population in the presence of guanidine hydrochloride (75 micrograms/ml) allowed selection of avirulent, guanidine resistant mutants with characteristics of t-, d-, DS+, DEAE-D+, G+. These mutants produced only small plaques (1.0-1.6 mm) and were designated PS-. The prototype and other selected strains that contained both large plaque and small plaque variants were characterized as PS+ and were invariably virulent. Although a variance in the d character has been demonstrated in some of the above strains, we conclude that virulence and the in vitro genetic markers t, d, DS, and DEAE-D are not related in our coxsackievirus B4 CD-1 mouse system. A relationship between plaque size (PS) and/or guanidine resistance (G+) of selected strains of coxsackievirus B4 and avirulence has been demonstrated in this system.

A simple method for typing clinical isolates of herpesvirus simplex: replication in the presence of 2H-1,3-oxazine-2,6 (3H)-dione (Oxauracil).

A rapid simple method for the typing of wild HSV isolates based on selective inhibition of HSV-2 replication by the pyrimidine analogue oxauracil (2H-1,3-oxazine-2,6(3H)-dione) has been developed. Chi-square analysis of the results of typing of 30 wild strains by serum neutralization tests, immunofluorescence, and oxauracil-inhibition indicates no significant differences in assignment of types to the isolates. The oxauracil-inhibition test obviates confusion of typing resulting from use of immune sera containing cross-reactive antibodies. We conclude oxauracil typing is a simple, rapid, and precise test which can be performed in any laboratory capable of isolating HSV.

Isolation of cytomegalovirus from cerebrospinal fluid of a congenitally infected infant.

The isolation of cytomegalovirus (CMV) from the CSF of an infant with congenital disease is documented. Samples of urine sediments and CSF were inoculated into tissue cultures of human foreskin fibroblast cells. Cytopathology was evident within 48 hours in those tubes inoculated with CSF. The urine cultures were later positive. All viral isolates were identified by complement fixation tests using known positive CMV antiserum.

Reactivity and antibody response to vaccination with bivalent influenza A/Victoria/75-A/New Jersey/76 vaccines in children with chronic pulmonary diseases.

Reactogenicity and antibody responses of high-risk children and adolescents (aged three to 18 years) to bivalent influenza A/Victoria/75-A/New Jersey/76 virus vaccine from nine participating centers were compared to the response of the total population of vaccinees in the multicenter study of the National Institute of Allergy and Infectious Diseases. Split-product vaccines given in two doses four weeks apart offered protection without a significant risk of side effects. Patients with chronic pulmonary disease did not differ from the total study population to antibody response or reactivity to vaccination with A/New Jersey strains.