Jay S. Epstein

Transfusion-transmitted babesiosis in the United States: summary of a workshop.

Infections of humans with intraerythrocytic parasites of the genus Babesia can be locally prevalent in diverse regions of the United States. Transfusion of blood and blood products collected from donors infected with Babesia may result in a serious illness that can be fatal. In September 2008, the Food and Drug Administration organized a public workshop to discuss the various aspects of transfusion‐transmitted babesiosis in the United States including the possible strategies to identify and defer blood donors who may have been infected with Babesia. Discussions were also held on the biology, pathogenesis, and epidemiology of Babesia species. In this article, we summarize the scientific presentations and panel discussions that took place during the workshop.

CONFERENCE REPORT: Transfusion-transmitted babesiosis in the United States: summary of a workshop

Infections of humans with intraerythrocytic parasites of the genus Babesia can be locally prevalent in diverse regions of the United States. Transfusion of blood and blood products collected from donors infected with Babesia may result in a serious illness that can be fatal. In September 2008, the Food and Drug Administration organized a public workshop to discuss the various aspects of transfusion‐transmitted babesiosis in the United States including the possible strategies to identify and defer blood donors who may have been infected with Babesia. Discussions were also held on the biology, pathogenesis, and epidemiology of Babesia species. In this article, we summarize the scientific presentations and panel discussions that took place during the workshop.

FDA approach to evaluation of pathogen reduction technology

athogen reduction (PR) is a novel approach to decreasing pathogen transmission by blood transfusion. It has the potential to inactivate a broad range of pathogens that could be found in donated blood products and thus it would complement existing methods to prevent or decrease the presence of pathogens. The current methods include donor selection, skin disinfection, diversion of initial collection, and testing for specific agents. The concept of PR holds great promise. Ideally, PR methods should inactivate all pathogens found in blood products, not damage the transfusion product, and be safe to be administered to all patients. In reality, PR methodology is in its infancy and has yet to reach its true potential. In their current form, PR treatments are unlikely to sterilize the transfusion product. This results from large loads of certain pathogens that could overwhelm the capacity of the treatment, resistant forms of pathogens, inaccessible pathogens due to interference from storage bag geometry, poor light energy delivery due to interfering substances, and the potential for human error during processing. The PR methods are not strictly pathogen specific and produce collateral damage to the transfusion products that is often evident by the decreased retention of these products in circulation. In addition, the toxicity of PR chemicals and their metabolites and adducts may not be realized until a large-scale patient population is exposed to them. The FDA will evaluate PR treatments for their efficacy in decreasing pathogens in blood products, their effect on the transfusion product, and their safety to the recipient of treated products.

Food and Drug Administration Conference on the Feasibility of Genetic Technology to Close the HIV Window in Donor Screening

ffective measures to reduce the transmission of viruses via blood and blood components have been implemented in recent years. Among these are doE nor education and deferral procedures and the use of screening tests. Despite these measures, the transmission of viral disease from units screened for antibody continues to occur, primarily as a result of donations in the antibody-negative, preseroconversion window period of infectivity. For human immunodeficiency virus (HIV), this residual risk is extremely small. Nonetheless, it remains the source of a major, ongoing public concern over the safety of the blood supply and tissue transplants. Because of recent improvements in antibody screening tests, and in light of the rapidly developing technology for detection of etiologic agents by genetic technologies, the Food and Drug Administration (FDA) thought it timely to reassess the HIV risk to the blood supply and to explore the potential of newer technologies to close the HIV window by direct detection of virus. Known methods for direct detection of virus include virus culture, p24 antigen, and viral nucleic acid testing. Virus culture is considered sensitive and specific, but labor-intensive and time-consuming. Viral p24 antigen tests are thought to be less sensitive and generally produce the same results as antibody tests in low-risk donors. Assays for detection of the viral genome, DNA and RNA, are considered to be highly sensitive but are still in the development and validation phase. Although several novel amplification approaches have been

Enterobacter cloacae Bloodstream Infections Traced to Contaminated Human Albumin

In August 1996, a patient in Kansas developed an Enterobacter cloacae bloodstream infection (BSI) shortly after receiving Albuminar, a brand of human albumin. Albuminar contamination was suspected. A case-control study of patients with primary gram-negative bacterial BSIs showed that patients with E. cloacae BSIs were significantly more likely than patients with non-E. cloacae gram-negative BSIs to have received Albuminar within 3 days of developing their BSIs (3 of 5 vs. 0 of 9; OR, undefined; P=.03). The E. cloacae isolate from the Kansas patient was found by pulsed-field gel electrophoresis to be identical to the isolate from the patients Albuminar vial, to isolates from 2 previously unopened Albuminar vials, and to an isolate from a Wisconsin patient who had received Albuminar. A worldwide recall of approximately 116,000 Albuminar vials took place. This multistate outbreak was detected because of clinical astuteness and prompt reporting. Combined epidemiological and laboratory approaches are valuable when investigating potentially contaminated blood components and plasma derivatives.

Rapid and sensitive detection of cell‐associated HIV‐1 in latently infected cell lines and in patient cells using sodium‐n‐butyrate induction and RT‐PCR

To develop a rapid and sensitive means of detecting cell‐associated human immunodeficiency virus (HIV), donor cells from HIV seropositive patients were treated with the potent viral activator sodium‐n‐butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT‐PCR). The sensitivity of RT‐PCR was estimated to be equivalent to 1 × 10−16 grams (0.1 fg) or approximately 64 copies of the input standard viral RNA per reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH‐2 and U1 cell lines were used as representatives of T‐lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT‐PCR assays. When peripheral blood mono‐nuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV‐positive PBMCs by RT‐PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT‐PCR intended to be used for detection of cell‐associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell‐associated viral load in vivo and without possible mutations brought about by long‐term co‐cultivation assays with cells from seronegative donors. J. Med. Virol. 52:179–189, 1997.

Inhibition of HIV replication by immunoliposomal antisense oligonucleotide

The sequence-specific suppression of HIV-1 replication using CD4 monoclonal-antibody-targeted liposomes, containing Rev antisense phosphorothioate oligonucleotides is described. Liposomes were prepared by encapsulating the 20-mer antisense DNA sequence of the rev HIV-1 regulatory gene, in the form of a phosphorothioate oligonucleotide. Specific targeting was accomplished by conjugating anti-CD4 mouse monoclonal antibody to the surface of the liposomes. HIV-1-infected H9 cells as well as peripheral blood T-lymphocytes were incubated with the immunoliposomes of antisense found to have potential antiviral effect. HIV-1 replication was reduced by 85% in antisense immunoliposome-treated H9 cells and peripheral blood lymphocytes, whereas the inhibition of HIV-1 replication was not observed using either empty immunoliposomes or immunoliposomes containing scrambled Rev phosphorothioate oligonucleotide sequences. The antiviral activity of both the free and the encapsulated oligonucleotides were assessed by p24, reverse transcriptase (RT) assays and polymerase chain reaction (PCR) analysis. Liposome preparations demonstrated minimal toxicity in H9 as well as in peripheral blood lymphocyte cell culture experiments. These in vitro culture results demonstrate the potential efficacy of immunoliposomes to inhibit HIV replication.

INTERFERON-GAMMA INHIBITS HIV-INDUCED INVASIVENESS OF MONOCYTES

HIV‐infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92‐kd metalloproteinase (MMP‐9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)‐infected monocytes with interferon‐γ at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP‐9 produced by HIV‐infected monocytes treated with interferon‐γ 1 day prior to infection with HIV as compared with that of untreated HIV‐infected monocytes. Monocytes treated with interferon‐γ 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP‐9 were substantially lower than those produced by monocytes inoculated with live HIV, heat‐inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat‐inactivated HIV did not result in increased invasiveness or high MMP‐9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4‐gp120 interactions and required active virus infection. Furthermore, addition of interferon‐γ to monocytes on day 10 after infection inhibited MMP‐9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon‐γ–induced down‐regulation of MMP‐9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN‐γ in monocytes. Down‐regulation of MMP‐9 in HIV‐infected monocytes by interferon‐γ may play an important role in the control of HIV pathogenesis.

Alternative strategies in assuring blood safety: An Overview

Assuring transfusion safety is an essential element of health care in all countries, requiring government commitment, national policy and a legal framework. Fundamental safety strategies include selection of low risk donors, Good Manufacturing Practices in preparation of blood components, and appropriate clinical use including avoidance of unnecessary transfusions. Hemovigilance, including surveillance for known adverse events and sentinel reporting of unexpected adverse events, enhances safety through benchmarking to promote best practices and by enabling rapid responses to new threats. Preventing transmission of infectious diseases is a principal safety concern. Selection of low risk donors includes use of screening questions to elicit risk factors known to be associated with transmissible infections. Laboratory testing for specific infectious disease markers is an established strategy for interdicting contaminated donations. The sensitivity, specificity, and operational convenience of laboratory testing have improved over time and newer technologies are imminent. Donor screening and laboratory testing, while highly effective in reducing risk, cannot eliminate all risk from known agents and must be developed de novo to address emerging infections. In contrast, pathogen reduction technologies offer the possibility for robust inactivation of a broad spectrum of blood transmissible agents and provide an added safeguard against newly emerging infectious threats of most types. Current pathogen reduction methods also inactivate leukocytes, adding safety benefits similar to leukocyte removal and product irradiation. However, to date, concerns about the safety and efficacy of cellular blood components treated by pathogen reduction have prevented approval of these technologies in the U.S. and Canada. FDA is promoting clinical and basic scientific studies to clarify these issues and would consider alternative approaches to assuring blood safety if pathogen reduction technologies are proven to be safe and effective.

Progress in monitoring blood safety.

I n this issue of TRANSFUSION, Zou and colleagues of the American Red Cross (ARC) report the marker prevalence and derive the incidence of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) for donations of approximately 66 million units of allogeneic whole blood collected between January 1999 and December 2008, encompassing the 10-year period since introduction of nucleic acid tests (NATs). The authors estimated the residual risk of these infections in donated blood as the product of incidence multiplied by the NAT window period for HIV and HCV. They provide an analysis of the temporal trends in risk and the underlying donor demographics. This article complements a previous publication by investigators at ARC and Abbott Laboratories in which the incidence and residual risk of hepatitis B virus (HBV) infection in whole blood donors was estimated by two independent methods for November 2006 through July 2008 and compared with a similarly derived result for November 1997 through July 1999. In a separate article in this issue, Zou and colleagues report on the marker rates, incidence, and residual risk of HIV, HCV, HBV, and human T-lymphotropic (HTLV) for apheresis donations of blood components from January 2004 through December 2008. They provide comparisons among these variables by apheresis component compared with whole blood collections. This report updates the findings of a previous investigation that was conducted under the first Retrovirus Epidemiology Donor Study (REDS-I) at five U.S. blood collection centers between 1991 and 1994. These studies represent the most current estimates of incidence and residual risk for the major bloodtransmissible viruses affecting the U.S. blood supply. Moreover, the analyses of temporal trends and demographics related to marker-positive donations provide insights into the sources of risk in the blood system and how they are changing over time. A few limitations warrant mention, however. First, it should be noted that the incidence estimates depend strongly on the number of NAT yield (RNA-positive, but antibody-negative) cases, which could have been affected by changes over time in the sensitivity of the NAT and serologic assays, as well as by current estimates for the marker-negative infectious window periods. Second, inferences of trends based on small numerators (number of incident cases) with very large denominators (total number of collections) can be misleading as to level of both statistical significance and public health importance. Third, it is possible that the data obtained solely within the ARC system might not be nationally representative. Nevertheless, these data provide us with a unique and very rich source of information that directs our attention appropriately to a number of issues and concerns. The role of NAT in enabling accurate assessments of donor incidence (number per 100,000 person-years) of HIV and HCV infection is central to these analyses. NATyield cases represent recently acquired and thus incident cases of infection, although estimation of the incidence rate in first-time donors still requires an extrapolation from the directly observed rate in repeat donors. In an examination of transfusion risk in the NAT era, it is perhaps also worth remembering that the introduction of NAT as a donor screening method for HIV resolved an otherwise vexing concern about disparate blood safety due to differences in sensitivity (and hence window period risk) of the available licensed screening tests for antibodies.