Gary E. Tegtmeier

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction

Among the three recently described GB viruses (GBV‐A, GBV‐B, and GBV‐C), only GBV‐C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV‐C proteins, the prevalence of GBV‐C RNA in human sera was studied using reverse transcription‐polymerase chain reaction (RT‐PCR). The prevalence of GBV‐C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV‐C was frequently detected in residents of West Africa, where the prevalence was >10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV‐C RNA. In addition, GBV‐C RNA sequences were detected in individuals diagnosed with non‐A‐E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV‐C RNA more than 1 year after the initial positive result.

Growth of bacteria in inoculated platelets: implications for bacteria detection and the extension of platelet storage

BACKGROUND: Recent reports from Europe have advocated the use of bacterial culturing of platelets on Day 2 or 3 of storage to extend the shelf life of platelets to 7 days, thereby reducing the outdating of platelets and preserving a limited medical resource. To assess the optimal timing, the necessary sensitivity, and the possible efficacy of bacterial detection, the bacterial growth characteristics were reviewed in 165 platelet units, each inoculated on the day of collection with one of the following organisms: Bacillus cereus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermidis from four previously published studies.

Monoclonal Antibody for Rapid Laboratory Detection of Cytomegalovirus Infections: Characterization and Diagnostic Application

Monoclonal antibodies to early (2H2.4, molecular weight 72,000 daltons) and late (2F3.0, molecular weight 68,000 daltons) antigens of the AD-169 strain of cytomegalovirus (CMV) were prepared by fusing mouse spleen cells with NS-1 mouse myeloma cells. The 2H2.4 monoclonal antibody produced a dense immunofluorescence with prominent lobular staining within the nucleus of CMV-infected substrate cells, whereas the reaction of 2F3.0 was more diffuse and generally involved the entire nucleus of the cells. Both monoclonal antibodies had little or no neutralizing activity against CMV in plaque-reduction assays. No cross-reactions were observed between these monoclonal antibodies and other members of the herpesvirus group. The 2H2.4 monoclonal antibody to early CMV antigen was used in a shell vial assay with a low-speed centrifugation step for the rapid (within 16 hours after inoculation) diagnosis of CMV infections. Optimal conditions for the test included centrifugation of shell vials at 700 X g for 45 minutes at 36 degrees C. An inoculum volume of 0.2 ml provided a reasonable balance between the optimal sensitivity for detecting specific viral fluorescence and the easy discrimination of the specific immunofluorescence from the background debris. Because of the commercial availability of the monoclonal antibody and the simplicity of the procedures used in the shell vial assay and subsequent fluorescence techniques, this rapid assay can be done in any laboratory that is familiar with cell culture manipulations.

CYTOMEGALOVIRUS: GENETIC VARIATION OF VIRAL GENOMES*

Genomic complexity of human CMV is one of the largest among various DNA viruses. With such genome complexity and widespread distribution, even a minimal frequency of mutation will be enough to create a complicated genetic heterogeneity in this virus. The virus genome is relatively stable during subcultivation in tissue culture. Variants with minor modification in restriction enzyme sites do gradually develop. These variants share substantial restriction fragment pattern homology with the parental virus. Virus DNA has a molecular weight of 150 X 10(6) daltons for a complete genome. The DNA contains 2 covalently linked L and S components which are separated by internal inverted repetitious sequences of both terminal-end sequences. L and S components are oriented invertably with 4 isomeric arrangements. No tandem repeated sequences have been found. Based on DNA restriction pattern analysis, we conclude that the majority of recurrent infections represent reactivation of existing latent viruses; however, reinfections by a new virus strain do occur occasionally. By studying virus strains isolated from the consecutive congenitally infected infants and strains from mothers and their congenitally infected offspring, we furthermore conclude that the latent virus in women is relatively stable genetically. Moreover, the virus strains after being transmitted to the offspring are still genetically similar to that of the mothers. As in vitro, spontaneous minor variations occur after the in vivo residence. In a long period of evolution the accumulation of minor variations might create great diversity with major similarity.

Frequency and duration of plasma CMV viremia in seroconverting blood donors and recipients

BACKGROUND : Both CMV‐seronegative blood and unscreened, filtered blood carry a low but definite risk of transmitting CMV infection. To explain this residual risk, evidence of cell‐free viremia was sought in seroconverting and seroprevalent blood donors and seroconverting transfusion recipients by means of a plasma‐based assay for CMV DNA.

Mumps Antibody Levels Among Students Before a Mumps Outbreak: In Search of a Correlate of Immunity

BACKGROUND In 2006, a mumps outbreak occurred on a university campus despite ≥ 95% coverage of students with 2 doses of measles-mumps-rubella (MMR) vaccine. Using plasma samples from a blood drive held on campus before identification of mumps cases, we compared vaccine-induced preoutbreak mumps antibody levels between individuals who developed mumps (case patients) and those who did not develop mumps (nonpatients). METHODS Preoutbreak samples were available from 11 case patients, 22 nonpatients who reported mumps exposure but no mumps symptoms, and 103 nonpatients who reported no known exposure and no symptoms. Antibody titers were measured by plaque reduction neutralization assay using Jeryl Lynn vaccine virus and the outbreak virus Iowa-G/USA-06 and by enzyme immunoassay (EIA). RESULTS Preoutbreak Jeryl Lynn virus neutralization titers were significantly lower among case patients than unexposed nonpatients (P = .023), and EIA results were significantly lower among case patients than exposed nonpatients (P = .007) and unexposed nonpatients (P = .009). Proportionately more case patients than exposed nonpatients had a preoutbreak anti-Jeryl Lynn titer < 31 (64% vs 27%, respectively; P = .065), an anti-Iowa-G/USA-06 titer < 8 (55% vs 14%; P = .033), and EIA index standard ratio < 1.40 (64% vs 9%; P = .002) and < 1.71 (73% vs 14%, P = .001). DISCUSSION Case patients generally had lower preoutbreak mumps antibody levels than nonpatients. However, titers overlapped and no cutoff points separated all mumps case patients from all nonpatients.

Transfusion‐Transmitted Cytomegalovirus Infections: Significance and Control

Abstract. Cytomegalovirus (CMV) is a herpes virus which can give rise to primary infections, reactivated infections, or reinfections in humans. Seroepidemiologic studies have shown CMV infection to be worldwide with the highest antibody prevalences detected in Third World countries; however, significant regional variations can be seen within a given country. Antibody prevalence varies directly with age and inversely according to socioeconomic status.

Performance of an algorithm for the reentry of volunteer blood donors deferred due to false-positive test results for antibody to hepatitis B core antigen

BACKGROUND: Blood donor testing for antibody to hepatitis B core antigen (anti‐HBc) has been used in the United States for more than 20 years as a surrogate to prevent transmission by transfusion of non‐A,non‐B hepatitis, as a human immunodeficiency virus surrogate, and to reduce transmission of hepatitis B virus (HBV). Nonspecific anti‐HBc assays have caused deferral of hundreds of thousands of otherwise qualified donors. A more specific anti‐HBc test and a sensitive HBV DNA test should permit donor reentry after false‐positive anti‐HBc.

Comparative yield of HCV RNA testing in blood donors screened by 2.0 versus 3.0 antibody assays.

BACKGROUND: Two HCV antibody tests (EIA 2.0 [EIA2], Abbott; and the Version 3.0 ELISA [EIA3], Ortho) are currently licensed for screening of US blood donors. Testing of donors for HCV RNA allows comparison of the sensitivities of the two antibody‐screening assays.

The use of a recombinant immunoblot assay in the interpretation of anti- hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors

ABSTRACT: Samples from prospectively followed recipients, their respective donors, and a cohort of random donors were used to evaluate the specificity and efficacy of a recombinant immunoblot assay (RIBA) as an adjunct to anti‐hepatitis C virus (HCV) testing by enzyme immunoassay (EIA). RIBA reacted (RIBA+) in 100 percent of patients who developed hepatitis associated with anti‐HCV seroconversion documented by EIA and in 100 percent of the EIA‐positive (EIA+) donors implicated in these cases. In contrast, RIBA reacted in none of 10 recipients who were EIA+ but did not develop hepatitis, in none of 7 EIA+ patients with hepatitis B or cytomegalovirus infection, in 33 percent of EIA+ donors who were not implicated in hepatitis transmission, and in 37 percent of EIA+ random donors. Hence, the vast majority of EIA+ individuals who have ancillary evidence of HCV infection react on RIBA, whereas the majority of EIA+ individuals in low‐risk settings do not react (RIBA− negative, or RIBA−). There was a strong association between RIBA reactivity and the presence of a surrogate marker (elevated alanine aminotransferase [ALT] and/or antibody to hepatitis B core antigen); 43 percent of RIBA+ implicated donors had a surrogate marker as compared to none of 14 EIA+, RIBA− donors. Among EIA+ random donors, 77 percent of those with a surrogate marker were RIBA+, as compared with 29 percent of those without a surrogate marker. In addition, in EIA+ donors, RIBA reactivity correlated with the extent of ALT elevation; 86 percent of those with an ALT > 135 IU per L were RIBA+ compared with 18 percent of those with an ALT < 30 IU per L. Among recipients of an EIAt, RIBAt blood unit, 73 percent developed hepatitisC and an additional 6 percent developed hepatitis, but were not positive for anti‐HCV. None o f 13 recipients of an E I A t, RIBA‐ unit developed hepatitis (p<0.00l). By using linked donor‐recipient sets, a RIBAt donor was found i n association with 75 percent of hepatitisC cases, which implies that these cases could have been prevented if anti‐HCV screening had been available at the time of the prospective study. Although considered supplemental rather than a confirmatory assay, RIBA correlates well with HCV infectivity and with surrogate markers of viral hepatitis. It sability to distinguish probable true‐positive E I A reactions from probable false‐positive reactions makes it a valuable adjunct t o blood donor noti fication, and it should be used prior to such notification whenever possible.