Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

BACKGROUND: Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. OBJECTIVE: To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. STUDY DESIGN: Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. RESULTS: A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. CONCLUSIONS: The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

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.

Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa.

Antisera were produced in mice immunized with 18 synthetic peptide conjugates representing various regions throughout the length of the outer membrane protein F molecule of Pseudomonas aeruginosa and analysed by flow cytometry to identify those antisera capable of binding to the surface of whole cells of P. aeruginosa. Antibodies to peptides 9, 18, 10, and 4 were significantly cell-surface reactive. The maximum median percentage of antibody-binding cells in this assay was 36.6%. Over six different determinations, peptide 9 antisera binding to the cells ranged from 16.9 to 57.0% of the cell population. We propose that the surface accessibility of protein F epitopes varies during the cell cycle.

Use of synthetic peptides to identify surface-exposed, linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa.

In a previous study (Hughes EE, Gilleland LB, Gilleland HE Jr. [1992] Infect Immun 60:3497-3503), ten synthetic peptides were used to test for surface-exposed antigenic regions located throughout the length of outer membrane protein F of Pseudomonas aeruginosa. An additional nine peptides of 11-21 amino acid residues in length were synthesized. Antisera collected from mice immunized with each of the 19 synthetic peptides conjugated to keyhole limpet hemocyanin were used to determine which of the peptides had elicited antibodies capable of reacting with the surface of whole cells of the various heterologous Fisher-Devlin immunotypes of P. aeruginosa. Cell surface reactivity was measured by an enzyme-linked immunosorbent assay (ELISA) with whole cells of the various immunotypes as the ELISA antigens and by opsonophagocytic uptake assays with the various peptide-directed antisera, immunotype 2 P. aeruginosa cells, and polymorphonuclear leukocytes of human and murine origin. Three peptides located in the carboxy-terminal portion of protein F elicited antibodies with the greatest cell-surface reactivity. Peptide 9 (TDAYNQKLSERRAN), peptide 10 (NATAEGRAINRRVE), and peptide 18 (NEYGVEGGRVNAVG) appear to have sufficient potential for further development as vaccine candidates for immunoprophylaxis against infections caused by P. aeruginosa. A topological model for the arrangement of protein F within the outer membrane of P. aeruginosa is presented.

Outer membrane protein F preparation of Pseudomonas aeruginosa as a vaccine against chronic pulmonary infection with heterologous immunotype strains in a rat model.

Outer membrane protein F (porin) was purified by extraction from polyacrylamide gels of cell envelope proteins of the Pseudomonas aeruginosa PAO1 strain. Rats were immunized intramuscularly with 25 micrograms of protein F on days 1 and 14 and then challenged on day 28 via intratracheal inoculation of bacterium-containing agar beads. On day 35 the lungs were either fixed for histological examination or submitted for quantitation of the bacteria present. protein F immunization afforded significant protection against challenge with six of six heterologous lipopolysaccharide immunotype strains of P. aeruginosa. By an enzyme-linked immunosorbent assay, the protein F-immunized rats had both immunoglobulin G and M antibody responses to cell envelopes of all six of the heterologous immunotype strains. Protein F immunization greatly enhanced the ability of the rats to clear the inoculated P. aeruginosa from the lungs and significantly reduced the incidence and severity of pulmonary lesions as compared with those in bovine serum albumin-immunized control rats. These data show the efficacy of outer membrane protein F as a protective vaccine in a rat model of chronic pulmonary infection.

Outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine against heterologous immunotype strains in a burned mouse model.

Outer membrane (OM) protein F (porin) was purified by extraction from polyacrylamide gels of cell envelope proteins of the Pseudomonas aeruginosa PA01 strain. Mice were immunized intramuscularly with 10 micrograms of protein F preparation on days 1 and 14 and then subjected to burn and challenge on day 28. Protein F immunization afforded significant protection above that provided by PA01 lipopolysaccharide (LPS) immunization against subsequent challenge with six of six heterologous LPS immunotype strains of P. aeruginosa. By an ELISA, the murine immune response revealed an IgG titer of 5,120 to protein F by day 30. Immunoblot analysis of antisera from protein F-immunized mice revealed bands with both protein F and protein H of cell envelopes of all immunotypes tested. Active immunization with OM protein H did not, however, afford significant protection to mice in this burned mouse model. These data show the efficacy of OM protein F as a protective vaccine in a murine model representative of human infection.