Transfusion-Transmitted Infections: an Update on Product Screening, Diagnostic Techniques, and the Path Ahead

ABSTRACT

The mandated testing of blood components for infectious diseases, to prevent transfusion-transmitted infections (TTIs), began in the 1950s. Since then, changes in predonation questionnaires and advances in testing techniques have afforded more sensitive and specific tests for pathogens, in addition to allowing earlier detection. Given that these approaches have very low but detectable failure rates, the recent development and implementation of proactive pathogen reduction approaches is the new forefront of TTI prevention strategies. With globalization and the ability of pathogens to evolve rapidly, continuous redefining of testing standards and laboratory techniques is paramount for maintaining a safe blood supply.

INTRODUCTION

Donor blood product safety and effectiveness are primary concerns of transfusion medicine specialists and blood collection centers worldwide. In the United States, prospective blood donors are first screened with a mandatory predonation questionnaire that was developed collaboratively by the blood collection industry and the Food and Drug Administration (FDA). Blood samples from donors who pass this step are then tested, using a growing list of highly sensitive infectious disease screening assays. Over the past several decades, advances in these laboratory testing techniques have allowed earlier detection of certain infectious diseases, resulting in a safer blood supply available to patients. However, emerging infectious diseases continue to pose a risk to the blood supply because of the considerable time that may be required to develop, to validate, and to gain regulatory approval for new testing methods to detect these agents. Thus, great efforts are being made to develop technologies to protect patients from new and emerging infectious agents for which we do not currently test. In this minireview, we describe the origins of blood supply infectious disease testing, advancements made to prevent transfusion-transmitted infections (TTIs), and future directions to improve the safety of donated blood components, with a primary focus on U.S. practices.

HISTORICAL OVERVIEW OF TESTING OF U.S. BLOOD PRODUCTS FOR INFECTIOUS DISEASES

The FDA is the regulatory body that oversees the infectious disease testing requirements for all blood components intended for transfusion. The FDA describes approved testing and defines TTIs in title 21, parts 610 and 630, of the Code of Federal Regulations, which has changed over the years as new infectious diseases emerge in the United States. In general, the FDA defines a TTI as a pathogen that is known to be fatal, to be life-threatening, or to cause severe impairment and that is potentially transmissible through the blood supply (1). Assessing donated blood products for antibodies to Treponema pallidum was the first TTI test to be required by the FDA, in the 1950s, following documented cases of syphilis infection associated with blood transfusion. Since that time, additional testing requirements have evolved following the identification of several additional infectious diseases found in highly transfused persons. In the 1960s, posttransfusion hepatitis (PTH) was found to have a strong association with the transfusion of blood products from paid donors (2). This revelation led not only to the development of the first assay for detecting the hepatitis B surface antigen (HBsAg) but also to the evolution of a volunteer-only blood donation system, both of which greatly reduced PTH rates. The finding that hepatitis B virus (HBV) could be transmitted by transfusion laid the groundwork for the subsequent discovery of non-A, non-B hepatitis, as neither hepatitis A virus nor HBV could account for all cases of PTH (3). Unfortunately, it would be two decades before hepatitis C virus (HCV) was cloned and identified as the causative agent; shortly thereafter, HCV antibody testing was implemented. Prior to the development of specific tests for HCV, surrogate markers associated with non-A, non-B PTH, such as alanine aminotransferase and anti-hepatitis B core antibody (HBcAb), were used to exclude donated blood products that carried a risk of transmitting PTH (4, 5). Testing for HBV and HCV now includes nucleic acid testing (NAT) for DNA and RNA, respectively, which further reduces the window period for detection of these viruses.

The AIDS epidemic was undoubtedly the greatest threat to the blood supply in the 20th century and was a major factor in how donated blood products are screened today. It has been estimated that approximately 12,000 people were infected with human immunodeficiency virus (HIV) via blood transfusions before 1985 (6). The hemophilia population suffered greatly from transfusion-transmitted HIV, with many patients becoming infected before the first case of AIDS was even documented (7). Fortunately, the discovery of HIV as the causative agent of AIDS and the relatively rapid implementation of antibody testing in 1985 led to a dramatic decrease in the number of transfusion-transmitted HIV cases. In addition, the FDA recommended changes to donor screening questionnaires to defer potentially HIV-infected persons from donating in the first place, by identifying behaviors associated with HIV/AIDS. Improvements in HIV-1 antibody tests and subsequent implementation of testing for anti-HIV-2, HIV-1 p24 antigen, and HIV RNA further reduced the number of cases of HIV transfusion transmission, by reducing the window period for detecting HIV infection in blood donors.

Human T-cell lymphotropic virus I (HTLV-I) and HTLV-II are retroviruses that were discovered before HIV (which was first designated HTLV-III). HTLV-I, which is endemic in Japan and the Caribbean region, is the etiological agent known to cause adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paresis (8). HTLV-II, which is endemic in the American Indian population, is closely related to HTLV-I, although its pathogenicity is less well understood (9). Despite the inherently lower prevalence of HTLV-I/HTLV-II infections in the United States, compared to areas in which the viruses are endemic, the FDA first recommended testing of all allogeneic blood donations for anti-HTLV-I/HTLV-II antibodies in 1988, after high rates of seroconversion were found following blood transfusion in areas in which the viruses are endemic (8, 10). Antibody testing specific for HTLV-II was introduced in the late 1990s, and the blood supply continues to be screened for both viruses today.

Insect-vector-transmitted infections are a constant threat to the global blood supply, and determining which pathogens to screen for in the United States can be challenging. Some carriers may be asymptomatic and unaware of being infected, adding to the difficulty of protecting the blood supply. Currently, the FDA recommends specific testing of donated blood products for Trypanosoma cruzi, West Nile virus (WNV), and Zika virus (ZIKV) (1). T. cruzi, the parasite responsible for Chagas disease, can cause chronic asymptomatic infection for decades before producing serious sequelae, such as cardiac and gastrointestinal complications. The number of cases of Chagas disease in the United States is estimated to be over 200,000; cases are largely seen among Latin American immigrants exposed to T. cruzi while living in areas in which the parasite is endemic (11). Rare reports of autochthonous cases have been documented, although the mechanism of disease acquisition is not fully understood (12). Since seroconversion in the United States is rare, the FDA recommends that all blood donors be tested only once for T. cruzi antibodies (13). The Flavivirus WNV first emerged in the United States in 1999 and was found to be transmissible through the blood supply via viremic donors, who might or might not have detectable WNV antibodies (14). The majority of individuals acutely infected with WNV are asymptomatic; however, serious neurological diseases can occur. Because anti-WNV antibody testing was determined to be insufficiently sensitive, a NAT method was developed and implemented in 2003 to screen all allogeneic blood donations (15). Another Flavivirus, ZIKV is endemic in areas of Africa and Asia and recently prompted global concern as spread of the virus reached Brazil by 2015 and the continental United States by 2016 (16). Similar to WNV, individuals infected with ZIKV are usually asymptomatic or present with nonspecific complaints, but serious neurological diseases, such as Guillain-Barré syndrome, have been associated with ZIKV infection (17, 18). The population at greatest risk for serious complications from ZIKV infection appears to be pregnant women. This concern came following the Brazilian outbreak and the association of ZIKV with devastating neurological effects, such as microcephaly, in fetuses and infants during acute maternal infection (19). Studies conducted at that time found that asymptomatic viremia could last from several weeks to several months after exposure and ZIKV RNA was prevalent in 1% of blood donors in Puerto Rico (20, 21). Therefore, in 2016 the FDA designated ZIKV a relevant TTI and recommended that all blood products intended for transfusion in the United States be tested for ZIKV RNA via NAT (22).

METHODS OF DONOR SCREENING AND TESTING

Screening of blood donors for infectious diseases consists of predonation questionnaires and postdonation testing of blood products. The donor history questionnaire assumes that donors understand the questions asked and that their answers regarding certain behaviors are truthful. Donor history questionnaires allow the collection center to assess the current general health of the donor, as well as certain behaviors associated with TTIs, prior to blood collection. Some medications and exposures elucidated through the questionnaire can result in the collection center deferring the donor either permanently or for a specified time period. The questionnaire can be helpful when laboratory testing is not available or feasible to detect certain infectious diseases that are identified as TTIs by the FDA (1). In the United States, the FDA requires blood collection centers to use predonation questions to assess donors for potential exposure to prion disease (classic Creutzfeldt-Jakob disease [CJD] and variant CJD [vCJD]), Plasmodium species, and Babesia species, due to the lack of FDA-approved screening tests (23–25). Donors are assessed for risks of classic CJD and vCJD and are excluded if they have a family history of CJD or potential exposure to CJD (e.g., dura mater graft or xenotransplantation) or vCJD (e.g., travel to Europe during an outbreak) (24). While cases of transfusion-transmitted CJD have never been documented in the United States, epidemiological studies indicate that vCJD has been transmitted via blood transfusions in the United Kingdom (26). There are currently no screening tests for CJD or vCJD that have been approved by the FDA, leading to the strict guidelines for deferring individuals with potential exposures.

Temporary deferrals exist for donors with recent travel to an area in which malaria is endemic, as well as for those who have emigrated from areas in which malaria is endemic (23). Transfusion-transmitted malaria in the United States is rare, and case numbers have declined in the past few decades with improvements in the donor history questionnaire. Reported cases are more commonly associated with immigrants from areas in which malaria is endemic, compared with travelers born in the United States, and Plasmodium falciparum is the most common species identified in such cases (27). The deferral periods for malaria, based only on the donor history questionnaire, have raised concerns for many years. Proposals have been made to implement testing of donors for Plasmodium species, so as not to lose potential noninfected donors during the deferral period and yet to exclude individuals who remain infectious beyond the deferral period (27). Unfortunately, a sufficiently sensitive malarial screening test for blood products has yet to be approved by the FDA.

Another parasite of concern for the United States blood supply is Babesia. Over 160 cases of transfusion-transmitted Babesia (mostly Babesia microti) have been documented since the 1970s, making it the most common transfusion-transmitted parasite in the United States (25, 28). The donor history questionnaire indefinitely defers donors who report a history of babesiosis, due to the severe and sometimes fatal disease that can occur following infection. Because asymptomatic carriers unaware of infection pose a threat to the blood supply, the AABB (formerly known as the American Association of Blood Banks) recommended that the FDA expedite approval of a Babesia blood donor screening test and that, when such a test is made available, it be applied to all red blood cell products, especially in areas with high levels of infection, such as the Northeast and upper Midwest (29). Indeed, in March 2018, the FDA licensed an antibody assay as well as a NAT to detect B. microti in whole blood; official FDA recommendations for screening of the blood supply are expected by the end of 2018. By design, the donor history questionnaire has allowed collection centers to select out donors with low risks of carrying infectious diseases. However, the questionnaire does not capture all infectious donors and, due to the seriousness of diseases caused by TTIs, testing methods must be highly sensitive and specific.

As an additional safeguard beyond the donor questionnaire, postdonation testing is utilized to detect some TTIs for which FDA-approved tests are available (e.g., HIV, HBV, HCV, HTLV-I/HTLV-II, ZIKV, WNV, T. cruzi, and syphilis). Testing methodologies for screening donated blood products for possible TTIs have vastly improved over the years. Blood collection centers must use highly sensitive, FDA-approved testing methods to screen for TTIs in donated blood products before making the blood products available for transfusion. Serological methods to detect antibodies have been the mainstay of TTI screening tests since syphilis testing was first implemented nearly 70 years ago. FDA-approved serology tests are currently utilized to detect HBV, HCV, HIV-1/HIV-2, HTLV-I/HTLV-II, syphilis, and T. cruzi. In general, if a donated blood product is found to be positive for a TTI, then that product is discarded and the donor is notified and deferred for a specified time period, depending on the TTI identified. Because false-positive results can occur with all highly sensitive screening tests, the FDA has established algorithms for confirmatory testing and reentry of individuals into the donor pool, again depending on the TTI in question.

The last documented case of transfusion-transmitted syphilis was over 50 years ago. This success is attributable to implementation of routine testing, improvements in the donor history questionnaire to defer donors with potential exposure, poor spirochete survival during blood product storage, and an overall decrease in the number of cases in the U.S. population (30). Potential blood donors are screened for syphilis with both the donor history questionnaire and serological testing of all blood donations. Individuals are deferred from donating if they report, on the questionnaire, a history of syphilis in the previous 12 months. Several FDA-approved treponemal and nontreponemal tests are available for screening donated blood products and confirming reactive results. Nontreponemal assays, such as the rapid plasma reagin (RPR) and the Venereal Disease Research Laboratory (VDRL) tests, are laborious and have the potential to defer noninfected donors due to a lack of specificity. In such cases, follow-up testing with a treponemal assay can rule out a false-positive result or confirm the initial positive result, with the latter resulting in a 12-month deferral (30). Treponemal tests, including enzyme immunoassays, agglutination assays, and fluorescent antibody assays, have better specificity and are favored by collection centers for their ability to be automated. An initial positive result from a treponemal screening assay can be confirmed with an alternative treponemal assay, with a second positive test result leading to deferral of the donor for 12 months (30).

Serological testing for HTLV-I/HTLV-II has evolved since screening was first implemented in the late 1980s. The first licensed serological assays were aimed at detecting HTLV-I but incidentally detected cross-reactive anti-HTLV-II antibodies, although with less sensitivity (31). Since those assays were unable to differentiate between the homologous HTLV-I and HTLV-II viruses, molecular testing was necessary to distinguish between the two for appropriate donor counseling, as the disease sequelae differ between these viruses (8). Unfortunately, such methods were neither widely available nor FDA approved for blood product screening. Therefore, improvements in the detection and differentiation of HTLV-II and HTLV-I were sought. In 1997, the FDA licensed an HTLV test with acceptable detection capabilities for both anti-HTLV-I and anti-HTLV-II antibodies and henceforth required all blood products intended for transfusion to be tested with this assay, permanently deferring donors found to be positive for HTLV-I or HTLV-II (31).

The first FDA-licensed T. cruzi serological screening assay for blood donors became available in 2007, followed by the 2010 FDA recommendation to screen all blood donors once for the parasite (13). A blood product found to be positive for T. cruzi in a screening test is removed from the blood supply, and the donor is notified and permanently deferred from donating blood. Supplemental licensed tests are available to rule out false-positive results. If individuals are found to be negative with a supplemental test, then they may be eligible for reentry into the donor pool after a 6-month period with negative results with two different screening tests, as well as a negative result with the supplemental test (13). In addition, the donor history questionnaire asks potential donors to report a history of Chagas disease, and those who respond yes are permanently deferred from donating blood. In the recently revised guidance, however, the FDA no longer recommends that this question be included, as current serological testing is sufficient for identifying T. cruzi among blood donors (32).

Serological screening tests have proved to be useful and cost-effective tools in helping to keep the blood supply safe. However, antibody development can take several weeks after pathogen exposure, resulting in a so-called window period between the time of infection and the point at which infectious donors can first be detected by testing. Indeed, much of the residual transfusion-related transmission of HIV, HCV, and HBV is from seronegative donors within this window period. During the 1990s, intense focus was placed on developing assays to identify these seronegative donors and to reduce the transmission rates of these pathogens further. NAT to detect viral DNA or RNA showed promising results, compared to antibody testing, by reducing the window period for HCV by 50 to 60 days and that for HIV by 11 to 15 days (33, 34). U.S. blood collection centers introduced HIV and HCV NAT screening for all donated blood products in 1999, with subsequent FDA approval of these testing methods in 2002. The NAT method detects both HIV-1 and HCV RNA and has similar sensitivities when used with individual donor samples or pools of 16 to 24 donor samples, which reduces overall test costs (34, 35). If a pooled sample tests positive, then subsequent testing is performed to determine the individual source of positivity. Once an individual donor tests positive for HCV or HIV via NAT, the blood product is excluded from the blood supply and the donor is notified and permanently deferred from donating blood products. In addition, the collection center performs a “look back” to identify any blood donations from that donor in the previous 12 months, removing those products from the blood supply or, if the unit has already been transfused, notifying the recipient of possible exposure. Because false-positive results can occur, there are specific scenarios in which donors with positive HCV or HIV NAT results are allowed to reenter the donor pool after a designated period (8 weeks for HIV and 6 months for HCV), if repeat testing shows negative antibody results and NAT results (34). Implementation of NAT has had a substantial effect in reducing the transfusion transmission of HCV and HIV to estimated residual risks of 1:1.2 million and 1:1.5 million, respectively (33, 35).

Detection of HBsAg and HBcAb were the first serological tests implemented for detection of HBV and are still used as part of the testing algorithm today. HBV NAT was added to the HCV/HIV testing platform in 2007, and the FDA formally recommended testing all blood products with HBV NAT in addition to HBsAg and HBcAb tests in 2012 (36, 37). Recent estimates predict a reduction in the window period for detecting HBV, compared to antibody testing, of 8 to 20 days (38). Individuals found to be positive for HBV are permanently deferred from donating blood. Similar to HIV and HCV NAT algorithms, individuals with suspected false-positive HBV NAT results may be eligible for reentry into the donor pool after a 6-month deferral period and subsequent negative HBcAb, HBsAg, and NAT results (36). HBV NAT has reduced the transfusion transmission to an estimated residual risk of approximately 1:1 million (38).

NAT is also used for the detection of WNV and ZIKV, as a measure to prevent transmission during the infectious acute viremic phase. As mentioned earlier, NAT for WNV began in 2003 and was initially performed on either individual samples or pools of 6 to 16 donor samples. However, concerns were raised after some studies found that testing of pooled samples was less sensitive than testing of individual samples, due to low-level viremia in donors, leading to breakthrough cases of transfusion transmission of WNV (15). Therefore, the FDA now recommends that blood products intended for transfusion must undergo individual NAT during times of high WNV infectivity (39). Blood products found to be positive for WNV are discarded, and the donor is notified and deferred for 120 days. Additionally, a look back is performed and any products from that individual donated in the previous 120 days that are still in inventory are also discarded (39). The FDA determined ZIKV to be a relevant TTI in 2016 and recommended that all blood products intended for transfusion be screened individually with an investigational ZIKV NAT (22). Deferral and look-back procedures for ZIKV are similar to those for WNV, as described above. The recommendations regarding testing of blood products for ZIKV are likely to change, as more information regarding disease pathogenesis and new models based on pooled NAT and seasonal testing results become available.

Another measure used to reduce transmission of infectious agents is leukoreduction, which removes leukocytes from donated cellular products (i.e., red blood cells and platelets) via a filter or through the blood collection process (i.e., in-process leukoreduction during apheresis collections). This procedure can reduce human leukocyte antigen (HLA) alloimmunization, febrile nonhemolytic transfusion reactions, and transfusion transmission of cytomegalovirus (CMV). The latter effect is important for protecting certain CMV-seronegative immunocompromised patients who could suffer significant morbidity and death during CMV infection. Deferring blood donors based on CMV status is not feasible, due to the high prevalence of CMV seropositivity in the general population. Leukoreduced blood products are considered by most to be CMV safe; however, large studies are lacking, making it difficult to develop formal guidelines regarding the use of products from CMV-seronegative donors versus CMV-untested, leukoreduced products for certain patient populations (40).

Bacterial contamination of blood products is less publicized than some of the TTIs discussed previously, but it accounts for far more complications of blood transfusion. The AABB and FDA have specific guidelines for preventing, detecting, and monitoring bacterial contamination in blood products, especially platelet products. Two types of platelet products are available in the United States, namely, whole-blood-derived platelets and apheresis-derived platelets. Whole-blood-derived platelets can be stored as single units or as pools of single units from 4 to 6 donors. Regardless of the platelet type, units are all stored for up to 5 days after collection at 20°C to 24°C, an ideal temperature for bacterial growth. In 2003, the AABB recommended multiple interventions to combat bacterial contamination, including proper skin cleansing, phlebotomy diversion kits, and implementation of bacterial culture techniques for all platelet components collected (41). Since the implementation of routine bacterial screening, it is estimated that 1 of 5,000 platelet units is contaminated with bacteria and septic transfusion reactions are associated with approximately 1 of 75,000 platelet transfusions (42). Although bacterial contamination is still a significant complication of blood transfusion, advancements in testing methods and pathogen reduction therapies are striving to improve the safety of the blood supply.

THE FUTURE OF BLOOD DONOR SCREENING AND EMERGING BLOOD SAFETY CONCERNS

Improvements in the donor history questionnaire and testing methodologies to screen blood donors have proved to be successful. Unfortunately, residual transmission of infectious agents still occurs, and current strategies do not protect the blood supply from new emerging pathogens. Therefore, a great deal of effort has been focused on developing an effective proactive approach that can be applied to all blood products, in hopes of reducing TTIs while minimizing the technical and financial burdens of screening individual blood donors. These methods, known as pathogen inactivation (PI) (43), have been used to reduce the transmission of pathogens in manufactured plasma derivatives (e.g., albumin and immunoglobulins) since the 1980s. Myriad techniques, including pasteurization, solvent-detergent treatments, low-pH incubation, and nanofiltration, have been used successfully to inactivate pathogens in plasma derivatives (44). These and other PI methods are widely used in Europe to treat platelet and plasma products intended for transfusion (45). However, the use of such methods to treat blood products in the United States has lagged in gaining FDA approval, pending large studies demonstrating an acceptable safety profile. Indeed, only recently were two PI methods approved for such use in the United States. In 2013, the FDA approved a solvent-detergent method, which inactivates enveloped viruses by disrupting the lipid membrane but is ineffective against nonenveloped viruses, for use on plasma products intended for transfusion. Newer PI methods include using chemical compounds to cross-link DNA or RNA, preventing replication of infectious agents. In 2014, a psoralen/UV-irradiation-based method was licensed by the FDA for treatment of plasma and apheresis platelet products intended for transfusion (46). PI methods using other compounds are currently being investigated for effectiveness and safety for the treatment of plasma, platelets, and red blood cells. Implementing such treatments may reduce the rates of residual TTIs and can potentially protect the blood supply from new emerging infections.

The HIV/AIDS epidemic of the 1980s first demonstrated the vulnerability of the blood supply to emerging pathogens. Since then, the AABB Transfusion Transmitted Diseases Committee continuously monitors and assesses the dangers of emerging unknown pathogens, as well as the geographical expansion of known pathogens. The combined efforts of this committee and the research community have allowed expeditious identification of emerging infectious diseases and implementation of screening methods to detect such pathogens. The response to the ZIKV outbreak of 2016 exemplifies the concerted efforts of these groups and their exceptional effects in protecting the blood supply. Since its inception, the AABB committee has published over 60 fact sheets on emerging infectious disease agents; it currently considers vCJD, dengue viruses, chikungunya virus, hepatitis E virus (HEV), and Babesia species as high priorities for increased monitoring in the U.S. blood supply (47, 48). With a multitude of infectious diseases and the increasing ease of human travel, concentrated efforts and a streamlined approach to detect and to eliminate pathogens from the U.S. blood supply are paramount.

SUMMARY

In this minireview, we have described the history of blood supply testing for infectious diseases, changes in predonation and postdonation testing aimed at reducing TTIs, and current investigations and developments to improve the identification and removal of infected donated blood components. PI shows great potential in reducing the transmission of pathogens in donated blood components; it would allow effective removal of infectious agents missed by current means of detection, as well as emerging infectious disease agents that are not currently tested for directly. This technology, along with AABB recommendations, is guiding the search for superior standards and methods to further reduce TTIs, thereby improving the safety of donated blood components and, in turn, patient care.