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. STUDY DESIGN AND METHODS: Quantitative culture data from inoculated platelet concentrates from five sites and four studies were combined into one database and analyzed for bacterial concentration thresholds (> or =10(1), > or =10(2), > or =10(3), > or =10(4), > or =10(5) CFU/mL) by day of storage. RESULTS: All examples of B. cereus, P. aeruginosa, K. pneumoniae, S. marcescens, and S. aureus had concentrations > or =10(2) CFU per mL by Day 3 after inoculation. By Day 4, all units with these organisms contained > or =10(5) CFU per mL. Units contaminated with S. epidermidis showed slower and more varied growth. By Day 3 after inoculation, 81.3 percent had 10(2) CFU per mL. By Day 4 after inoculation, 46 (95.8%) of 48 units had concentrations > or =10(2) CFU per mL. CONCLUSION: These experiments suggest that an assay capable of detecting 10(2) CFU per mL on Day 3 of storage would detect the vast majority of bacterially contaminated platelet units, prevent many cases of platelet-associated bacterial sepsis, and provide a scientific basis for the extension of the current platelet storage time. It would be expected that a rare, slow-growing organism could escape such a detection scheme.

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.

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 greater than 135 IU per L were RIBA+ compared with 18 percent of those with an ALT less than 30 IU per L.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative prevalence and risk factors of HTLV-I and HTLV-II infection in US blood donors.

The clinical significance of human T-cell lymphotropic virus type II (HTLV-II) infection, unlike that of HTLV-I, is unknown, and the major known association of HTLV-II seropositivity is with intravenous drug abuse. Screening of blood donors for HTLV-I, now routine in North America, does not distinguish this retrovirus from HTLV-II. To find out more about the seroepidemiology of and risk factors for HTLV I and II, blood from 480,000 volunteer donors in five geographically separate US urban centres was tested for antibodies to HTLV-I/II and HIV-1. Confirmed HTLV-I/II seropositive donors were then followed up by DNA amplification to distinguish type I from type II and by interviews focusing on possible risk factors. HTLV seroprevalence was 3.3 times greater than that for HIV-1 (0.043% vs 0.013%). DNA amplification on 65 of the 207 HTLV-I/II seropositive donors revealed that 34 (52%) had HTLV-II infection and 28 (43% had HTLV-I; 3 samples were uninformative. Interviews of 49 donors showed that whereas HTLV-I was principally associated with donor origin from endemic regions, the major risk factor for HTLV-II infection was intravenous drug use. The surprisingly high rate of HTLV-II infection in US blood donors raises important public health and donor counselling issues since HTLV-I infection is associated with adult T-cell leukaemia and a neurological disorder while the pathogenicity of HTLV-II is as yet unclear.

Use of TrpE/Gag fusion proteins to characterize immunoreactive domains on the human immunodeficiency virus type 1 core protein.

The human immunodeficiency virus (HIV) p24 core protein is one of the most immunogenic of HIV structural proteins. Infected individuals develop high titers of antibodies against p24 early in infection, which makes anti-p24 antibodies important serological markers. However, despite the clinical importance of the anti-p24 response, no systematic study to characterize the antigenic domains on the p24 protein has been reported. We report here on the use of 12 overlapping fragments of the HIV type 1 p24 protein, synthesized in bacteria as TrpE/Gag fusion proteins, to identify at least two and possibly three antigenic domains on the p24 protein. In addition, we note that different HIV-seropositive sera exhibited different patterns of reactivity with the p24 domains presented on our fusion proteins.

Posttransfusion cytomegalovirus infections.

Prospective studies of transfused immunocompetent patients in the 1960s and early 1970s showed high rates of cytomegalovirus (CMV) infection, ranging from 16% to 67%. Similar studies conducted in the 1980s have reported rates of infection of 1% in seronegative patients. Little morbidity or mortality has been associated with these CMV infections. In contrast, transfusion-transmitted CMV infections in seronegative immunocompromised premature infants and bone marrow transplant recipients have resulted in significant morbidity and mortality. Blood transfusion is a less important risk factor in recipients of solid organ transplants. The risk of infection, however, can be reduced by providing blood from CMV-seronegative donors or frozen deglycerolized red blood cells. Leukocyte removal by filtration or centrifugation is also likely to reduce the risk. Prophylaxis with CMV immune globulin can ameliorate the severity of posttransfusion-posttransplant CMV infections.

Cytomegalovirus and blood transfusion.

The problem of transfusion-transmitted cytomegalovirus (CMV) infection differs from that for other transfusion-transmitted infections in that only patients who are immunocompromised require CMV-free blood or components. The virus is cell-associated and transmission appears to be due to reactivation of latent virus in white blood cells. As a herpes virus, CMV can be responsible for primary infections, reactivations or reinfections in humans. The use of restriction endonuclease techniques is sometimes necessary to pinpoint the origin of infections. Serological studies have shown that CMV infection is worldwide, but seropositivity rates vary widely being highest in underdeveloped countries, rising both with age and lower socio-economic status. Provision of CMV seronegative blood therefore involves considerable administrative as well as laboratory effort and planning, especially if panels of previously tested, seronegative donors are organized. Serious complications of transfusion-transmitted CMV infection (which can occasionally prove fatal) are only seen with immuno-suppressed patients (commonly low birth weight infants or transplant recipients). Prevention or amelioration of CMV infection with appropriate patients can be attempted by reducing the number of white blood cells present in blood or components by filtration or washing, administration of CMV immune globulin or provision of blood found by serological screening to be CMV-seronegative.

Comparison of two testing methods to determine hepatitis B surface antibody response to hepatitis B vaccine among health-care workers.

Because of variations in reported seroconversion rates and to compare enzyme-linked immunoassay (EIA) and radioimmunoassay (RIA) methods to assess hepatitis B vaccine response, hepatitis B surface antibody (anti-HBs) was tested in 116 of 174 high-risk hospital employees enrolled in a hepatitis B vaccine program. All individuals were vaccinated with three injections of Heptavax-B, 1.0 ml containing 20 micrograms of HBsAg intramuscularly in the deltoid. The same lot of appropriately stored vaccine was used. Of the 41 individuals tested within zero to six months postvaccination, 35 (85%) and 38 (93%) were positive by EIA and RIA, respectively. Of the 75 individuals tested 7-24 months postvaccination, 61 (81%) and 71 (95%) were positive by EIA and RIA, respectively. Results of EIA tests performed at two laboratories were similar. Of 109 individuals positive by RIA, 13 did not have protective antibody levels. In contrast, of 96 individuals positive by EIA, only one did not have a protective antibody level. RIA may be a more sensitive test for anti-HBs, but a positive EIA result correlates better with protective antibody levels.

AIDS retrovirus antibodies in hemophiliacs treated with factor VIII or factor IX concentrates, cryoprecipitate, or fresh frozen plasma: prevalence, seroconversion rate, and clinical correlations.

Antibodies to the AIDS retrovirus, specifically to human T cell lymphotropic virus, type III, and AIDS-associated retrovirus, were detected with increasing prevalence in a population of 190 hemophiliacs from western Pennsylvania between 1981 and 1984: 7.7% in 1981, 20.0% in 1982, 45.5% in 1983, and 62.5% in 1984. The seropositive included approximately three fourths of those receiving factor VIII concentrate, nearly one third of those receiving factor IX concentrate, nearly one fifth of those receiving cryoprecipitate, and none of those receiving fresh frozen plasma. The seroconversion rate, determined on 43 seropositive hemophiliacs from this group who were serially sampled, was 0% in 1977, 4.7% in 1978, 4.9% in 1979, 2.6% in 1980, 10.5% in 1981, 52.9% in 1982, 87.5% in 1983, and 100% in 1984. Of 27 seropositive for three or more years (since 1982 or before), four (15%) have developed AIDS and seven (26%), diffuse lymphadenopathy (ARC); of 16 seropositive for less than three years, none has developed AIDS and three (19%) have developed ARC. The mean time from seroconversion to onset of ARC, 0.8 +/- 0.2 years (SEM), was shorter (P less than .001) than the time to onset of AIDS, 4.1 +/- 0.6 years. These findings confirm the widespread presence of AIDS retrovirus and support the association of these retroviruses with the acquired immunodeficiency syndrome and related conditions.

Transfusion-transmitted cytomegalovirus infections: significance and control.

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. Numerous prospective studies of blood transfusion recipients carried out since 1966 have shown marked differences in infection rates but relatively little associated disease. Infection rates were highest in seronegative recipients given large amounts of fresh blood. Recently published reports have shown substantially lower infection rates than earlier studies, a change likely to be due to the current practice of transfusing fewer units of older blood. CMV has not been found to play a significant role in the etiology of posttransfusion hepatitis. CMV infections have been found to be an important source of morbidity and mortality in immunocompromised patients. Several studies of transfused, premature infants have shown significant differences in infection rates and disease expression. Seronegative low-birth-weight infants receiving blood from seropositive donors are at greatest risk. Blood from CMV-seronegative donors substantially lowers the risk of infection. Receiving a kidney or heart from a CMV-seropositive donor appears to be a more salient risk factor than blood transfusion in renal and cardiac transplant patients who are also more likely to have symptomatic CMV infections. Leukocyte transfusions have been found to be a significant source of CMV infection and disease in bone marrow transplant patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Early and late antigens of human cytomegalovirus: electroimmunodiffusion assay of numbers, relationships, and reactivities with donor sera.

Early antigens (EA) of human cytomegalovirus extracted from cytosine arabinoside-blocked cells infected with 0.01-20 infectious units (IU)/cell were assayed with human serum by electroimmunodiffusion (EID). The number of detectable EA types increased from one to eight as the IU/cell was raised from 0.01 to 10. There was no increase in the number of EA with further increases in IU/cell, with prolonged culture, or when detergent was included in the extraction buffer. At least five of the eight EA gave reactions of identity with late-time antigens (LTA) extracted from unblocked cells at late times postinfection. In studies on a panel of sera from donors who were excreting virus and donors who were not, EID was as sensitive as conventional techniques (complement fixation and indirect hemagglutination for LTA, indirect immunofluorescence for EA) in detection of both types of antibodies from excretors but less sensitive in not detecting low levels of the antibodies in some of the sera from nonexcretors. No consistent relationships were observed between donor virological status and the numbers or types of antibodies to EA and LTA.

Relation of titers of antibodies to CMV in blood donors to the transmission of cytomegalovirus infection.

Titers of antibody to cytomegalovirus (CMV) of 529 persons whose blood had been supplied to 51 selected patients who underwent open-heart surgery were determined by indirect hemagglutination (IHA) and IgM-specific indirect immunofluorescence (IFA). Twenty-eight patients showed evidence of active CMV infection after transfusion (seroconversion or a fourfold rise in titer by IHA), whereas 23 showed no serological change. Patients with active CMV infections had received, on average, a greater number of blood units (12.9 vs. 7.9), of which more were seropositive (6.9 vs. 3.5), than did patients who showed no serological change. Those seropositive units of blood that had been transfused into the group that showed evidence of active infection, however, had a lower geometric mean titer than did those transfused into the group that showed no serological change (1:654 vs. 1:1,360). Seven (1.3%) of the 529 blood donors had CMV-specific IgM titers (by IFA) of greater than or equal to 1:16; each of the seven recipients of their blood subsequently showed evidence of active CMV infection. This study suggests that donor blood with high IHA titers may prevent transmission of CMV infection, whereas blood from donors with IgM antibody to CMV may transmit CMV.