Factors in enhancing blood safety by nucleic acid technology testing for human immunodeficiency virus, hepatitis C virus and hepatitis B virus.

In the last few decades through an awareness of transfusion transmitted infections (TTI), a majority of countries have mandated serology based blood screening assays for Human immunodeficiency virus (HIV), Hepatitis C virus (HCV), and Hepatitis B virus (HBV). However, despite improved serology assays, the transfusion transmission of HIV, HCV, and HBV continues, primarily due to release of serology negative units that are infectious because of the window period (WP) and occult HBV infections (OBI). Effective mode of nucleic acid technology (NAT) testing of the viruses can be used to minimize the risk of TTIs. This review compiles the examples of NAT testing failures for all three viruses; analyzes the causes for failure, and the suggestions from retrospective studies to minimize such failures. The results suggest the safest path to be individual donation testing (ID) format for highest sensitivity, and detection of multiple regions for rapidly mutating and recombining viruses. The role of blood screening in the context of the donation and transfusion practices in India, the donor population, and the epidemiology is also discussed. World wide, as the public awareness of TTIs increases, as the recipient rights for safe blood are legally upheld, as the possibility to manage diseases such as hepatitis through expensive and prolonged treatment becomes accessible, and the societal responsibility to shoulder the health costs as in the case for HIV becomes routine, there is much to gain by preventing infections than treating diseases.

Molecular methods and platforms for infectious diseases testing a review of FDA-approved and cleared assays.

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

Prevalence and quantitation of parvovirus B19 DNA levels in blood donors with a sensitive polymerase chain reaction screening assay.

BACKGROUND: Blood donor parvovirus B19 DNA prevalence with sensitive nucleic acid test assays has recently been demonstrated to be higher than that found with assays designed to detect high viral titers in the plasma manufacturing sector. STUDY DESIGN AND METHODS: Stored plasma aliquots from 5020 donations collected between 2000 and 2003 at seven US blood centers were tested. Testing was performed with a real-time B19 DNA polymerase chain reaction (PCR; TaqMan, Applied Biosystems) assay with a 50 percent limit of detection (LOD) of 1.6 IU per mL (95% confidence interval [CI], 1.2-2.1 IU/mL) and a 95 percent LOD of 16.5 IU per mL (95% CI, 10.6-33.9 IU/mL). Confirmation and quantitation of B19 DNA was accomplished by retesting of two additional subaliquots. Confirmed-positive specimens were tested for the presence of anti-B19 immunoglobulin M (IgM) and IgG with FDA-licensed assays. RESULTS: B19 DNA prevalence was 0.88 percent (95% CI, 0.64%-1.2%). Among the 23 donations with B19 DNA titers of at least 20 IU per mL, the median DNA concentration was 105 IU per mL with an interquartile range of 42 to 481 IU per mL; the highest value was 1869 IU per mL. All B19 DNA-positive donations were positive for the presence of IgG and 10 (23%) were also positive for the presence of IgM; IgM seropositivity was associated with increasing DNA levels (p = 0.0013). CONCLUSION: Low-level B19 DNA was detected in nearly 1 percent of donations. The 23 percent of DNA-positive donations with both IgM and IgG B19 antibody most likely represent acute resolving infection, whereas those with IgG but no IgM are most consistent with a more chronic and possibly persistent phase of B19 infection.

Screening the blood supply for West Nile virus RNA by nucleic acid amplification testing.

BACKGROUND: The use of nucleic acid amplification tests of "minipools" of 16 samples to screen blood donors for West Nile virus RNA began in July 2003. We report the yield and characteristics of positive donations and the incremental yield and safety of nucleic acid amplification tests of individual donations. METHODS: Reactive minipools were analyzed to identify the individual reactive donations. For the regions with the highest yield on minipool testing, retrospective nucleic acid amplification testing was performed on individual donations that were negative on minipool testing. Reactive donations were confirmed by alternative nucleic acid amplification tests and IgM and IgG tests, and donors were followed to document seroconversion. RESULTS: From July 1 through October 31, 2003, 677,603 donations were prospectively screened for West Nile virus by minipool testing, yielding 183 confirmed viremic donations (0.027 percent, or 1 in 3703 donations). Retrospective individual testing of 23,088 donations from high-prevalence regions that were negative on minipool testing yielded 30 additional units with a low level of viremia, with 14 additional viremic units detected by prospective testing of individual donations late in the 2003 transmission season. Of all the viremic units detected, 5 percent were detected only by individual testing and were negative for IgM antibody, 29 percent were detected by individual testing after IgM seroconversion, and 66 percent were detected by minipool testing. West Nile virus infection was confirmed in both recipients of IgM-negative units that were reactive on individual testing, whereas neither recipient of antibody-positive blood components that were reactive on individual testing was infected. In 2004, prospective testing of individual donations in regions that yielded donations that were reactive on minipool testing resulted in a 32 percent incremental yield of units with a low level of viremia that would have been missed by minipool testing. CONCLUSIONS: Although nucleic acid amplification testing of minipools of blood donations prevented hundreds of cases of West Nile virus infection in 2003, it failed to detect units with a low level of viremia, some of which were antibody-negative and infectious. These data support the use of targeted nucleic acid amplification testing of individual donations in high-prevalence regions, a strategy that was implemented successfully in 2004.

Prospective evaluation of community-acquired acute-phase hepatitis C virus infection.

BACKGROUND: More than two-thirds of hepatitis C virus (HCV) infections in Western countries are caused by injection drug use, but prospective clinical data regarding the most common mode of HCV acquisition are rare, in part because acute-phase HCV infection is usually asymptomatic. METHODS: To characterize acute-phase HCV infection, 179 HCV antibody-negative injection drug users were prospectively evaluated; 62 (34%) of these patients had seroconverted. Twenty of the participants who seroconverted had long-term follow-up with consistent monthly sampling before and after seroconversion, allowing detailed study. RESULTS: The first indication of HCV infection was the presence of HCV RNA in serum, which preceded elevation of alanine transaminase levels and total bilirubin levels to > or =2 times baseline in 45% and 77% of patients, respectively. No subjects had jaundice. The median time from initial viremia to seroconversion was 36 days (range, 32-46 days). In one instance, viremia was detected 434 days before seroconversion. However, in no other case was HCV RNA detected >63 days before seroconversion. In subjects with viral persistence, a stable level of HCV RNA in the blood was noted in some subjects within 60 days after the initial detection of viremia, but in others, it was not apparent until >1 year later. In subjects with long-term viral clearance, HCV became persistently undetectable as early as 94 and as late as 620 days after initial viremia. CONCLUSIONS: These data underscore the importance of nucleic acid screening of blood donations to prevent HCV transmission and of long-term follow-up to ascertain whether there is viral persistence, at least among injection drug users.

Assessment of the target-capture PCR hepatitis B virus (HBV) DNA quantitative assay and comparison with commercial HBV DNA quantitative assays.

Recent clinical studies suggest that hepatitis B virus (HBV) load and genotype may be independent predictors of responses to antiviral therapies. However, it is difficult for clinicians to accurately determine viral loads in patient samples because results--both the values and the units of measure--can vary greatly among different tests. Accordingly, the World Health Organization (WHO) has produced the first international standard for HBV DNA for nucleic acid amplification technology (NAT) assays. In the present study, we describe the performance of the target-capture PCR HBV DNA quantitative assay for the quantitation of HBV DNA in clinical samples and reference panels. The range of quantitation was between 50 and 10(10) IU/ml. The sensitivity and accuracy of the target-capture PCR assay were demonstrated by using the HBV panel from Quality Control for Medical Diagnostics (QCMD) and the WHO HBV DNA standard. The target-capture PCR assay quantitated the six genotype A members of the QCMD panel and dilutions of the WHO HBV DNA standard within an accuracy of 74 to 142%. Compared to current serological methods, the assay offers window period reductions of 19 days prior to HBV surface antigen and 26 days prior to HBV e antigen detection. The target-capture PCR assay was also compared with four commercially available NAT assays, and the various units of measure were standardized with respect to the international units of the WHO HBV DNA standard. The target-capture PCR assay is a sensitive, accurate, high-throughput, rapid, and reproducible assay for the determination of HBV loads.

Detection and quantitation of HBV DNA in the WHO International Standard for HIV-1 RNA (NIBSC code: 97/656).

Nucleic-acid amplification technology (NAT) assays have been implemented for HCV and HIV-1 in the United States, and many parts of Europe, Australia and Asia. Nucleic acid detection assays utilize many different technologies, and the WHO International Standards for nucleic acid tests are widely used to compare them. Currently, several laboratories are developing an assay for simultaneous detection of HCV RNA, HIV-1 RNA and HBV DNA. In the course of such development it was observed that the WHO International Standard for HIV-1 RNA (97/656) was positive for HBV DNA. In this report we confirm the presence of HBV DNA in the HIV-1 international standard through the qualitative Procleix-Ultrio assay. Further, using the TaqMan technology, through quantitative Bead Capture-TaqMan assay, we report that approximately 1000IU/ml dilution of HIV RNA contains approximately 4500IU/ml of HBV DNA.

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana.

The risk of hepatitis B virus (HBV) transmission by transfusion in sub-Saharan Africa is considered to be relatively low, and testing of blood donors is often not done or is done relatively poorly. To re-examine this attitude, we identified HBV chronically infected blood donors from a major hospital in Ghana with a range of hepatitis B surface antigen (HBsAg) assays. Test efficacy was estimated using HBV DNA as a gold standard, and the risk of HBV infection in blood recipients was estimated for different testing strategies. Particle agglutination, dipstick, and enzyme immunoassay (EIA) HBsAg screening detected 54%, 71%, and 97% of HBV infectious donors, respectively. The risk of HBV transmission to recipients less than 10 years old ranged between 1:11 and 1:326 with blood unscreened and screened by EIA, respectively. For older recipients, the risk decreased a further 4-fold because of the high frequency of natural exposure to HBV. A total of 98% of HBsAg-confirmed positive samples contained HBV DNA. HBV DNA load was less than 1 x 10(4) IU/mL in 75% of HBsAg-reactive samples, most of them anti-HBe reactive. Approximately 0.5% of HBsAg-negative but anti-HBc-positive samples contained HBV DNA. The use of sensitive HBsAg tests is critical to prevent transfusion transmission of HBV infection to young children in a population with a 15% prevalence of chronic HBV infection in blood donors. However, this will not have much effect on the prevalence of this infection unless other strategies to protect children from infection are also advanced in parallel.