Trypanosoma cruzi screening in Texas blood donors, 2008-2012.

Chagas disease is an important emerging disease in Texas that results in cardiomyopathy in about 30% of those infected with the parasite Trypanosoma cruzi. Between the years 2008 and 2012, about 1/6500 blood donors were T. cruzi antibody-confirmed positive. We found older persons and minority populations, particularly Hispanic, at highest risk for screening positive for T. cruzi antibodies during routine blood donation. Zip code analysis determined that T. cruzi is associated with poverty. Chagas disease has a significant disease burden and is a cause of substantial economic losses in Texas.

Reacting to an emerging safety threat: West Nile virus in North America.

West Nile virus (WNV) entered North America in 1999 and in 2002 was shown to be transfusion transmitted. With competent mosquito and bird vectors throughout the United States and Canada, WNV clinical disease continues at epidemic proportions. Due to these facts, blood donor screening was implemented prior to the 2003 mosquito season and occurs using a variety of strategies. A combination of minipool (MP) nucleic acid amplification testing (NAT) during the " non-season, " coupled with the conversion to the more sensitive individual donation (ID) NAT in epidemic locations during epidemic times, has been successful in detecting approximately 1500 infected blood donors. Assuming that each donation was infectious and manufactured into 1.45 blood components, testing has therefore prevented close to 2200 recipient infections and potential clinical disease. During this same time, transfusion transmission has occurred from seven MP NAT-nonreactive/ID NAT-reactive units (6 in 2003 and 1 in 2004), or a total of 30 transfusion transmitted cases since WNV has been identified in North America. Because WNV occurs in infected blood donors at low concentrations (i.e., lower viral loads than HIV or HCV with the highest viral load of 580,000 copies/mL observed in a blood donor), a trigger strategy that is used in most of the US consisting of two NAT-reactive donations detected by MP NAT and a frequency of 1:1000 or greater has been developed. Since the full implementation of the MP to ID NAT trigger strategy, there have been no documented WNV transfusion transmissions. Because WNV is an acute infection that only occurs seasonally, other strategies have been proposed, such as seasonal testing, which has been implemented successfully in Canada (Quebec), coupled with a screening question used in the " non-season " of whether the donor has been in the US during the past 56 days; if so, WNV NAT is performed. WNV is an example of an emergent agent in which a rapid series of interventions has been successful in controlling transmission through blood transfusion.

NAT update: where are we today?

The US blood supply has been tested for HIV-1 and HCV using Nucleic Acid Amplification Testing (NAT) of small pools since early in 1999. Since the implementation of NAT under an investigational new drug (IND) application, the results for yield and false positivity have been amazingly consistent for greater than two years of testing even among multiple programmes using two different test methodologies and manufacturers: Gen-Probe/Chiron transcription-mediated amplification (TMA) and Roche PCR. All programmes in the US and Canada use NAT as a criterion for cellular as well as frozen product release. The focus of this manuscript is to provide an update of the programmes in the US and Canada, provide data in support of p24 antigen replacement by HIV-1 NAT, and discuss the projections of residual risk of HIV, HCV and HBV following NAT and the associated cost/benefit.

Risk of hepatitis and retroviral infections among blood donors and introduction of nucleic acid testing (NAT).

Blood donors represent a carefully selected population of individuals. In particular, they have reduced prevalence and incidence rates for hepatitis and retroviral markers when compared to the general population. Serological testing of all donations further decreases the risk of collecting an infectious blood unit to negligible levels. Nevertheless, a significant fraction of this residual risk will be eliminated by the introduction of nucleic acid testing for viral genomic material in donor samples. This testing, which has already reached the initial phases of implementation, will be performed on pooled samples, at least in the first instance.

Current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections.

BACKGROUND: In August 2009, a group from the AABB (Stramer et al., Transfusion 2009;99:1S-29S, Emerging Infectious Disease Agents and their Potential Threat to Transfusion Safety; http://www.aabb.org/resources/bct/eid/Pages/default.aspx) published a Supplement to Transfusion that reviewed emerging infectious disease (EID) agents that pose a real or theoretical threat to transfusion safety, but for which an existing effective intervention is lacking. The necessary attributes for transfusion transmission were outlined including: presence of the agent in blood during the donor's asymptomatic phase, the agent's survival/persistence in blood during processing/storage, and lastly that the agent must be recognized as responsible for a clinically apparent outcome in at least a proportion of recipients who become infected. Without these attributes, agents are not considered as a transfusion-transmission threat and were excluded. Sixty-eight such agents were identified with enough evidence/likelihood of transfusion transmission (e.g., blood phase) and potential for clinical disease to warrant further consideration. In the Supplement, Fact Sheets (FS) were published providing information on: agent classification; disease agent's importance; clinical syndromes/diseases caused; transmission modes (including vectors/reservoirs); likelihood of transfusion transmission, and if proven to be transfusion-transmitted, information on known cases; the feasibility/predicted success of interventions for donor screening (questioning) and tests available for diagnostics/ adapted for donor screening; and finally, the efficacy, if known, of inactivation methods for plasma-derived products. The Supplement included a separate section on pathogen reduction using published data. Agents were prioritized relative to their scientific/epidemiologic threat and their perceived threat to the community including concerns expressed by the regulators of blood. Agents given the highest priority due to a known transfusion-transmission threat and severe/fatal disease in recipients were the vCJD prion, dengue viruses and the obligate red-cell parasite that causes babesiosis (B. microti and related Babesia). Although the focus of the Supplement was towards the United States and Canada, many of the agents (and the process) are applicable worldwide. NEXT STEPS: Since the publication of the Supplement, six new FSs (yellow fever viruses-including vaccine breakthrough infections, miscellaneous arboviruses, XMRV, human parvoviruses/bocaviruses other than B19, and most recently the Middle East respiratory syndrome coronavirus, MERS-CoV) were added and 14 existing FSs updated (Anaplasma, Babesia, Bartonella, Erhlichia, chronic wasting disease-CWD, human prions other than vCJD, vCJD, Coxiella burnetii-the agent of Q fever, dengue viruses, HAV, HEV, Japanese encephalitis-JE complex, tick-borne encephalitis viruses-TBEV, and human parvovirus B19). Also, tables were released outlining pathogen reduction clinical trials/results (published) and availability/commercial routine use of such technologies by country. Of necessity, the list of EID agents is not, and can never be, complete due to the nature of emergence. We recognized that a system of assessing the risk/threat of EIDs for their potential impact on blood safety and availability must include processes for monitoring, identifying, evaluating, estimating severity, assessing risk and developing interventions. Thus, a 'toolkit' containing the necessary 'tools' from EID monitoring (horizon scanning) to validation/effectiveness evaluations of interventions is being developed. The goal is, to develop a systematic approach to risk assessment and intervention development for the impact of emerging infectious upon blood safety intended to educate and provide advise about risks/interventions in a timely/accurate fashion. CONCLUSIONS: The process and final product (toolkit) including methods to monitor EID agent emergence, identification/recognition of a transfusion-transmission threat, methods for quantitative risk assessments, and the appropriate management of such threats should be considered for implementation by all blood systems.

In vitro evaluation of the protective role of human antibodies to West Nile virus (WNV) produced during natural WNV infection.

BACKGROUND: West Nile virus (WNV) is endemic in the United States and transmissible by transfusion. Since 2003, the US blood supply has been screened by nucleic-acid tests (NAT) for WNV in minipools (MP-NAT) of 6 or 16 specimens. WNV infection begins with low-level viremia detectable only by individual testing (ID-NAT) and no detectable WNV antibodies. Viremia then increases to levels detectable by MP-NAT, and antibodies become detectable; later, viremia decays to levels detectable only by ID-NAT before becoming undetectable. All but 1 documented WNV transmission by transfusion involved blood components negative for WNV antibodies, raising the question whether WNV antibody-positive blood components with low levels of WNV RNA are infectious. METHODS: Specimens from 102 viremic donors with and without WNV antibodies were used to investigate infectivity in cultures of Vero cells and human monocyte-derived macrophages (MDMs). RESULTS: In Vero cell culture, 54 (74%) of 73 WNV antibody-negative specimens and 10 (36%) of 28 WNV antibody-positive specimens were infectious. In a random subset of 20 specimens tested in MDM culture, 7 (88%) of 8 WNV antibody-positive specimens and 12 (100%) of 12 WNV antibody-negative specimens were infectious. CONCLUSION: WNV antibodies do not always protect susceptible cells from WNV infection in vitro. RNA positivity in the presence of antibody cannot be ignored as a theoretical risk for blood recipients and needs further investigation.

Antibodies to a novel antigen in acute hepatitis C virus infections.

BACKGROUND: Conformational viral proteins potentially play an important role in the immunobiology of acute hepatitis C virus (HCV) infection and may enable earlier antibody detection. MATERIALS AND METHODS: HCV RNA was detected using nucleic acid testing. Early antibody production was evaluated using three enzyme immunoassays (EIAs) containing antigenic proteins not present in licensed EIAs. Respectively, these contained: (1) multiple-epitope fusion antigen (MEFA) 7.1-NS3/4a, (2) F and Core, and (3) E1/E2 proteins. NS3/4a is a conformational antigen retaining protease and helicase enzymatic activities. MEFA 7.1 contains the linear epitopes used in licenced EIAs, including the latest EIA-3.0, in combination with genotype 1-3 specific epitopes. Forty-two RNA positive, EIA-3.0 negative samples, including two persistently serosilent cases, were used to evaluate these research EIAs. As controls, 54 EIA-3.0 negative/RNA negative and three HCV RNA+/antibody positive specimens were included. RESULTS: Only the MEFA 7.1-NS3/4a EIA was positive in seven (17%) of the 42 HCV RNA + specimens, in all three EIA-3.0 positive controls but in none of 54 EIA-3.0 negative/HCV RNA negative controls. Notably, six of the seven (86%) specimens had evidence of active hepatitis (ALT > 210 IU/l). The two serosilent cases were research EIA negative. CONCLUSION: A novel EIA with conformational and linear epitopes detected HCV antibodies in 17% of viraemic specimens missed by the standard reference EIA-3.0. Our research EIA appears to detect HCV antibodies closer to the initiation of acute hepatitis. Given that the average RNA-positive, antibody-negative window period is 56.4 days, this 17% yield would translate into a 10-day earlier detection of antibodies.

Self-reported symptoms associated with West Nile virus infection in RNA-positive blood donors.

BACKGROUND: In 2003, West Nile virus (WNV) nucleic acid amplification testing (NAT) was implemented to detect potentially infected donors. Of more than 5.3 million donations screened prospectively by the American Red Cross during the epidemic periods of 2003 and 2004, 974 were NAT-reactive and 519 confirmed-positive. A subset of both the confirmed-positive and the false-positive groups was assessed for demographic characteristics, symptoms, and symptom reporting relative to date of donation. STUDY DESIGN AND METHODS: All donors with initial WNV NAT-reactive results were invited to participate in a study that included a demographic, symptom, and date-of-symptom questionnaire. WNV confirmed-positive cases were compared to false-positive controls for comparison of frequency of symptom reporting before, on the day of, and after donation. RESULTS: Enrolled cases and controls were similar in all characteristics except cases were more likely to live in rural areas. Symptoms were reported by 61 percent of cases versus 20 percent of controls, with 74 percent of symptoms reported by cases within the 14 days after donation. The frequency of headache and fever reported together in the 7 days before donation was not significantly different between cases and controls; only the individual frequencies of headache, eye pain, and new rash during this time were significantly different. The most commonly reported symptoms, after adjustment for symptom reporting by controls, were headache, new rash, and generalized weakness; these symptoms were reported by 25 percent of cases. CONCLUSIONS: The demographic characteristics of infected donors reflected the rural nature of the 2003 to 2004 WNV epidemics. This study suggests that asking donors about predonation headache and fever had no detectable contribution to blood safety.

Effectiveness of human T-lymphotropic virus (HTLV) recipient tracing (lookback) and the current HTLV-I and -II confirmatory algorithm, 1999 to 2004.

BACKGROUND: This study reports on the efficacy of an investigational human T-lymphotropic virus (HTLV)-I and -II lookback program in the context of differing confirmatory testing algorithms. STUDY DESIGN AND METHODS: The results of testing approximately 35 million donations for anti-HTLV-I and -II were evaluated for two recent periods reflecting the use of two different confirmatory algorithms. The number of seroconverting donors was established for the entire period, and the results of lookback on their prior donations were investigated. RESULTS: The dual enzyme immunoassay (EIA) strategy was used throughout both study periods and resulted in a 57 to 76 percent reduction in the number of samples requiring confirmatory testing. From May 2000 to February 2002, a total of 9138 samples were repeatedly reactive by the primary screening test; of the concordant EIA-reactive samples, 461 (12%) were confirmed by Western blot, whereas 3083 (79%) were indeterminate. From March 2002 to December 2004, a total of 21,291 samples were repeatedly reactive; of the concordant EIA-reactive samples, 1099 (22%) were confirmed by the State of California's reference laboratory and only 273 (5%) were equivocal. Overall, 38 or 1 in 921,000 donations were from a seroconverting donor with 32 prior donations within the lookback period. Of those 32, components from only 11 were transfused to recipients who survived; of these, 4 were tested and all were nonreactive for HTLV-I and -II antibodies. CONCLUSION: Use of creative algorithms can increase the efficacy of anti-HTLV-I and -II confirmatory testing and reduce the number of indeterminate results. Currently, lookback for HTLV-I and -II has a very low yield, and its public health benefit is questionable.

Performance of ORTHO HCV core antigen and trak-C assays for detection of viraemia in pre-seroconversion plasma and whole blood donors.

OBJECTIVE: Logistics and cost of nucleic acid amplification testing (NAT) screening preclude its current use in many developing countries. Development of hepatitis C virus (HCV) core antigen assays offer an alternative to NAT. We evaluated two specimen populations to assess the sensitivity, relative to NAT, of the HCV core antigen (HCVcAg) ELISA (enzyme-linked immunosorbent assay) test system and the trak-C assay: (1) plasma donor HCV NAT-conversion panels and (2) cross-sectional whole blood donor NAT yield specimens. METHODS: Differential sensitivities among NAT (NGI; Chiron/Gen-Probe) and both HCVcAg assays (Ortho-Clinical Diagnostics, Rochester, NY) were evaluated using: (1) 102 serial ramp-up phase specimens from 37 plasma donor NAT-conversion panels (Alpha Therapeutic/BioClinical Partners); and (2) 42 cross-sectional whole blood donor NAT yield specimens (confirmed RNA positive, antibody negative) plus 54 NAT false-positive specimens (American Red Cross). RESULTS: Viral load among the plasma donor NAT-conversion panels at the cutoffs for HCVcAg and trak-C assays were 32 000 copies/ml (95% confidence interval [CI] 8000-120 000) and 8000 copies/ml (95% CI: 2200-28 000), respectively. The mean (95% CI) difference in window period reduction compared to routine mini-pool NAT screening (estimated sensitivity 100 copies/ml) was delayed 5.2 days (2.2-7.6 days) for HCVcAg assay and 3.8 days (2.1-5.5 days) for the trak-C assay. Among the 42 NAT yield specimens, the HCVcAg assay detected 31 (74%) as core antigen-positive while the trak-C assay detected 37 (88%) as core antigen-positive. Viral loads for the five specimens not detected by the trak-C HCVcAg assay ranged from 100 to 7770 copies/ml. All 54 NAT false-positive specimens were non-reactive on both HCV core antigen assays. CONCLUSION: These data indicate that the trak-C assay has sensitivity approaching routine mini-pool NAT screening for the detection of seronegative HCV infection. In the absence of routine NAT screening for early HCV infection, the use of an HCV core antigen assay should be considered.

Presumptive identification of Klebsiella pneumoniae on m-FC medium.

Klebsiella pneumoniae is a human and animal pathogen frequently encountered in the environment. The membrane-filtration method utilizing m-FC medium for the detection of fecal coliforms yields a colony type easily distinguishable as K. pneumoniae. These colonies appear as atypical light blue, nucleated, mucoid colonies differing considerably from the typical dark blue fecal coliform colonies.

US NAT yield: where are we after 2 years?

The US blood supply has been tested for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) using nucleic acid amplification testing (NAT) of pools of small number of samples since early 1999. Since the implementation of NAT under an investigational new drug (IND) application, the results for the yield and false positivity have been remarkably consistent for greater than 2 years of testing even among multiple programmes using two different test methodologies and manufacturers: Gen-Probe/Chiron transcription-mediated amplification (TMA) and Roche polymerase chain reaction. All programmes in the US and Canada use NAT as a criterion for cellular as well as frozen product release. The focus of this paper is to provide an update of the programmes in the US and Canada, provide data in support of p24 antigen replacement by HIV-1 NAT and discuss the projections of residual risk of HIV, HCV and hepatitis B virus (HBV) following NAT and the associated cost/benefit.

Nucleic acid testing for transfusion-transmissible agents.

Pooled nucleic acid testing for HIV and hepatitis C virus (HCV) has been successfully implemented in the United States. Reactive rates for nucleic acid testing (NAT) are comparable to those for serological testing. The combined yield for HIV and HCV RNA confirmed-positive, seronegative donations for the 1st year of testing has been 62 HCV NAT-reactive donations among more than 16.3 million screened (1:263,000) and four HIV NAT-reactive, p24 antigen-negative donations among greater than 12.6 million screened (1:13,150,000). The observed yield of NAT confirmed-positive donations is compatible with estimates derived from window-period projections and recent measurements of incidence. Of the HCV NAT-reactive donations identified, one has not sero-converted over a 300-day period. The predominant recent risk factor identified for HCV NAT-reactive donors is intravenous drug use.

Septage treatments to reduce the numbers of bacteria and polioviruses.

Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended.

Improved growth of Thermus aquaticus on cellular lysates.

Studies on the thermophilic bacterium Thermus aquaticus have demonstrated the ability of this organism to exist on its own cell lysates. The use of 0.1% tryptone, and 0.1% yeast extract and a mineral salts lysate medium increased growth 9-to 44-fold over the above medium without cell lysates. It has also been established in this study that, due to the high growth temperature of T. aquaticus, its metabolic activities are greatly accelerated resulting in early cell destruction. Therefore, bacterial lysates from which other cells could derive nutrients and/or growth factors would constantly be available in the environment immediately around the organism to aid the growth of cells in natural as well as man-made thermal environments.

Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population.

BACKGROUND: There has been continuing progress in measures to reduce the risk of transfusion-transmitted infection, including introduction of serologic tests of increased sensitivity and the recent implementation of investigational NAT in small pools of samples. STUDY DESIGN AND METHODS: Data relating to all blood donations to the American Red Cross have been consolidated into a single database. The prevalence of confirmed-positive test results for HBsAg, HCV, HIV, and HTLV were evaluated for each year for first-time donors from 1995 through 2001. Incidence rates for these infections were evaluated among repeat donors having at least two donations in a 2-year period. The frequencies of HIV-1 RNA- and HCV RNA-positive, seronegative donations were assessed for first-time and repeat donations. The relationship risk = (window period) x (incidence) was used to assess residual risk among repeat donations and to evaluate the incidence of HCV and HIV infection among first-time donors. RESULTS: During the study period, prevalence rates for all markers declined significantly over time: in 2001, the rates per 100,000 were 75.6 for HBsAg, 299 for HCV, 9.7 for HIV, and 9.6 for HTLV; the corresponding incidence rates (/100,000 person-years) were 1.267, 1.889, 1.554, and 0.239, respectively. Estimates of residual risk in donations from repeat donors (after NAT) for HCV and HIV were 1 per 1,935,000 and 1 per 2,135,000, respectively. However, incidence rates for these agents are approximately two times greater among first-time donors. For both HCV and HIV, NAT yield was concordant with that predicted by current window-period models. CONCLUSION: These data cover about half of all the whole blood collected in the United States. They suggest increasing improvement in transfusion safety and clearly define the benefit of pooled NAT.

The cost-effectiveness of NAT for HIV, HCV, and HBV in whole-blood donations.

BACKGROUND: The risk of viral infection associated with blood transfusion is lower than ever before because of aggressive screening and testing practices. NAT technology has lowered that risk even further but at an additional cost to the health-care system. STUDY DESIGN AND METHODS: Marginal cost-effectiveness of NAT for HIV, HCV, and HBV in whole-blood donations was calculated with a previously published Markov decision model. This model was updated with disease incidence data from all 2001 American Red Cross whole-blood donations as well as window-period data from the Retrovirus Epidemiology Donor Study (REDS). RESULTS: Whole-blood donation NAT for HIV and HCV is expected to cost between 155 US dollars million (minipool NAT) and 428 million US dollars(single-donation NAT) per year in the US and avert 4 to 7 HIV infections and 56 to 59 HCV infections. Adding HBV NAT would be expected to avert 9 to 37 HBV infections at an additional cost of between 39 million US dollars and 130 million US dollars per year. Overall, NAT would cost between 4.7 million US dollars and 11.2 million US dollars per quality-adjusted life-year saved. Discontinuing HIV p24 antigen and HBc testing would offset this somewhat. CONCLUSIONS: The cost-effectiveness of whole-blood NAT is poor. The testing cost would need to decrease significantly to bring the cost-effectiveness in line with most other accepted medical practices.

Risk factors for HCV infection among blood donors confirmed to be positive for the presence of HCV RNA and not reactive for the presence of anti-HCV.

BACKGROUND: In 1999, NAT of blood donations was implemented to detect "window-period" infections. Blood donors who have confirmed NAT results positive for the presence of HCV in the absence of anti-HCV are likely to have been recently infected. Of over 26.8 million donations tested between March 3, 1999, and March 31, 2003, 810 were HCV-reactive by NAT. A subset of these donors was assessed for recent exposure risk. STUDY DESIGN AND METHODS: All anti-HCV- blood donors with reactive, unconfirmed HCV NAT results were invited to participate in a study that included an extensive demographic and risk questionnaire. Confirmed HCV+ cases were compared to HCV- (falsely positive) controls for histories of potential risk factors during the 6 months before donation. RESULTS: Recent injection drug use (IDU) was independently associated with HCV infection (29.2% vs. 0% of cases vs. controls, p < 0.001). In addition, likely sources were identified for three other cases (4.6%), including occupational exposure, sexual contact with an HCV-infected partner (who was an IDU), and perinatal exposure, none of which was known to the donors at the time of donation. Incarceration was independently associated with HCV infection among the group not reporting IDU and after removal of the three donors with likely sources of risk (14.6% vs. 1.3% of cases vs. controls, p < 0.001). CONCLUSIONS: A likely risk, primarily IDU, was found for 43 percent of HCV+ donors whose infections were identified solely by NAT. Because the maximum efficiency of the donor history questions may have been reached, NAT will continue to be an important measure to interdict recently infected blood donors.