Nucleic Acid Amplification Technology Testing Research Articles

Introduction of nucleic acid amplification technology for blood screening in Hungary

Background & AimsWith this study, we intended to report our experiences about introducing nucleic acid testing for blood donation screening for human immunodeficiency virus, hepatitis B and C in Hungary. This study summarizes the experiences of nucleic acid amplification technology (NAT) screening of the first 16 months.MethodsThe study was conducted from 5 June 2018 to 30 September 2019 in two phases. During the first 7 months of the study, the NAT tests were performed in parallel with serology. The yields of the methods were compared. After the initial seven months, only seronegative samples were tested by NAT. NAT tests were performed in minipools of 96.ResultsIn this 16‐month period, nearly 400 000 donations were screened by NAT. During the first phase, almost 150 000 samples were tested in parallel by serology and NAT screening. NAT screening during Phase I failed to identify 4 HCV‐ and 1 HBV‐reactive donations. In the second phase, about 245 000 seronegative samples were screened, with only one HBV reactive yield.ConclusionsNucleic acid amplification technology testing proven to be an optimal diagnostic adjunct to serological screening, since it can help to identify early window period infections and occult hepatitis B infections. According to our experience however, the used pooling strategy represents a dilution rate of nucleic acid targets, which makes identifying these types of infections difficult. According to our cost–benefit analysis, reducing the pool size to 60 would result in a 60% sensitivity increase, at the expense of increasing the daily costs by 25%.

Proceedings of the Food and Drug Administration public workshop on pathogen reduction technologies for blood safety 2018 (Commentary, p. 3026).

On November 29, 2018, experts in the field of infectious diseases, pathogen reduction technologies (PRTs) and other participants from blood centers, academia, and industry gathered at the Food and Drug Administration (FDA) White Oak Campus in Silver Spring, Maryland, for a 2‐day public workshop entitled “Pathogen Reduction Technologies for Blood Safety.” The workshop opened with welcome remarks from Dr. Nicole Verdun, Director, Office of Blood Research and Review (OBRR), Center for Biologics Evaluation and Research (CBER), FDA, followed by introductory remarks from Dr. Peter Marks, Director, CBER, FDA. The first day of the workshop focused on blood‐borne infectious agents and their impact on blood safety, experiences of the American Red Cross, and other blood establishments in implementing FDA‐approved pathogen inactivation (PI) technology for plasma and platelets (PLTs) in the United States and novel PRTs under consideration for whole blood (WB) and red blood cells (RBCs). The second day opened with welcome remarks from Dr. Chintamani Atreya, Associate Director for Research, OBRR, CBER, FDA. The focus was on emerging innovations relevant to PRTs and potential alternatives to PRTs. The workshop concluded with remarks on insights for future research and development in this area for blood and blood product safety from infectious agents. A brief introduction of each session by the session moderator followed by a summary of the speaker presentation as submitted by the moderator and speaker are reported here.

Assessment of HIV transfusion transmission risk in South Africa: a 10-year analysis following implementation of individual donation nucleic acid amplification technology testing and donor demographics eligibility changes.

In 1998 we estimated that 34/million infectious window period donations were entering the blood supply at the South African National Blood Service. Selective use of donations based on donor race-ethnicity reduced this risk to 26/million donations but was deemed unethical. Consequently, in 2005 South African National Blood Service eliminated race-ethnicity-based collection policies and implemented individual-donation nucleic acid testing (ID-NAT). We describe the change in donor base demographics, human immunodeficiency virus (HIV) detection rates, and transfusion-transmissible HIV risk. In ten years 7.7 million donations were tested for anti-HIV and HIV RNA. Number of donations, HIV prevalence, ID-NAT yield rate, serology yield rate and residual transfusion-transmissible HIV risk were analyzed by donor type, race-ethnicity, age, and sex. Multiple regression analysis was performed to investigate the determinants of HIV-positive and nucleic acid testing yield donations. The combined strategy of increasing donations from black donors and implementing ID-NAT increased the proportion of donations from black donors from 6% in 2005 to 30% in 2015 (p < 0.00001), and reduced the transfusion-transmissible risk from 24 to 13 per million transfusions. ID-NAT interdicted 481 (1:16,100) seronegative window period donations, while one transfusion-transmissible case (0.13 per million) was documented. Race-ethnicity and donor type were highly significant predictors of HIV positivity, with adjusted odds ratio for first-time donors of 12.5 (95% confidence interval, 11.9-13.1) and for black race-ethnicity of 31.1 (95% confidence interval, 28.9-33.4). The proportion of serology yields among HIV-infected donors increased from 0.27% to 2.4%. ID-NAT enabled the South African National Blood Service to increase the number of donations from black donors fivefold while enhancing the safety of the blood supply.

Application of combined nucleic acid test with serologic test in blood screening for voluntary blood donors in Weifang City of Shandong Province

Objective To explore the combined application of nucleic acid test and serologic test in blood screening for voluntary blood donors in Weifang City of Shandong Province. Methods From June to December 2016, a total of 55 852 voluntary blood donors in Weifang Blood Centre were selected as study subjects. Enzyme-linked immunosorbent assay (ELISA) was used to perform serologic tests on plasma specimens from all voluntary blood donors. ELISA tests included HBsAg, anti-hepatitis C virus (HCV), anti-human immunodeficiency virus (HIV), anti-Treponema pallidum (TP) and alanine aminotransferases (ALT). For the plasma specimens with negative results of ELISA test, the nucleic acid amplification technology (NAT) was used to detect the viral nucleic acid by viral nucleic acid detection system of Kehua Bio-engineering Co., Ltd of Shanghai and viral nucleic acid detection system of Novartis Diagnostics of Switzerland. NAT tests included HBV DNA, HCV RNA and HIV RNA. For the plasma specimens with HBsAg negative of ELISA test and HBV DNA positive of NAT split test and identification test, the five viral markers test of hepatitis B virus (HBV) were performed, which included HBsAg, HBsAb, HBeAg, HBeAb and HBcAb test. And according to the results of five viral markers of HBV, the HBV infection of voluntary blood donors were presumed. χ2 test was used to compare positive detection rates of nucleic acid in plasma specimens of Kehua viral nucleic acid detection system and Novartis viral nucleic acid detection system. The continuous correction χ2 test was used to compare the effective split rate of Kehua viral nucleic acid detection system and the positive detection rate of identification test for HBV DNA, HCV RNA and HIV RNA in plasma specimens of Novartisc viral nucleic acid detection system. Results ① In 55 852 plasma specimens from voluntary blood donors, 1 962 plasma specimens were positive for ELISA test. In 53 890 plasma specimens with negative ELISA results, 19 (0.035%, 19/53 890) plasma specimens were positive for NAT test. ② There were 53 890 plasma specimens were performed NAT test in this study. Among them, the positive detection rate of nucleic acid in plasma specimens of Kehua viral nucleic acid detection system was 0.014% (4/28 199), the positive detection rate of nucleic acid in plasma specimens of Novartis viral nucleic acid detection system was 0.058% (15/25 691). There was a statistically difference between the positive detection rate of Kehua viral nucleic acid detection system and that of Novartis viral nucleic acid detection system (χ2=7.50, P<0.05). ③ The effective split rate of Kehua viral nucleic acid detection system was 44.4% (4/9). The positive detection rate of identification test for HBV DNA, HCV RNA and HIV RNA in plasma specimens of Novartisc viral nucleic acid detection system was 50.0% (15/30). There was a statistically difference between the effective split rate and identification test positive detection rate (χ2=0.03, P=0.87). ④ There were 18 voluntary blood donors who were negative for HBsAg of ELISA test and positive for HBV DNA of NAT test. There were 5 serologic infection patterns in these 18 voluntary blood donors, including occult HBV infection accounted for 88.9% (16/18), and suspectedwindow periodHBV infection accounted for 11.1% (2/18). Conclusions There is a certain risk of missed detection of ELISA serologic tests in blood screening for voluntary blood donors. NAT test is an effective supplement to ELISA. The mixed sample test reduces the positive detection rate of the viral nucleic acid, by reducing the sensitivity of NAT test. The risk of transmitting HBV through blood transfusion needs to be highly vigilant for the blood collection and supply institution in Weifang City. Nucleic acid test as a supplemental method for serological test can further reduce the risk of transmitting HBV through blood transfusion, and ensures blood safety. Key words: Blood safety; Nucleic acid amplification techniques; Serologic tests; Enzyme-linked immunosorbent assay; Blood donors; Blood screening

Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi‐dye nucleic acid amplification technology screening test

The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads <12IU/ml. The cobas(®) TaqScreen MPX test, version 2.0, has an advantage over the current Roche blood screening test, the cobas TaqScreen MPX test, for screening donations in countries with a high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0.

Evaluation of two, commercial, multi‐dye, nucleic acid amplification technology tests, for HBV/HCV/HIV‐1/HIV‐2 and B19V/HAV, for screening blood and plasma for further manufacture

The cobas TaqScreen MPX Test, version 2.0, a multiplex, multi-dye nucleic acid amplification technology (NAT) test from Roche was evaluated by two European Blood Banks, the German Red Cross Blood Donor Service, Frankfurt, Germany and Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain. In addition, the cobas TaqScreen DPX Test was evaluated for the simultaneous detection and quantitation of parvovirus B19 and the detection of hepatitis A virus (HAV). The performances of the two tests were evaluated regarding the analytical sensitivity, the reproducibility of the tests using samples containing low concentrations of each virus and cross-contamination using samples containing high titres of virus. The analytical sensitivity of the MPX Test, version 2.0, obtained by the German Red Cross Blood Donor Service was 1·1, 3·9 and 43·3 IU/ml for HBV, HCV and HIV-1, respectively. The comparable analytical sensitivity at Centro de Hemoterapia y Hemodonación de Castilla y León was 3·5, 17·6 and 50·6 IU/ml for HBV, HCV and HIV-1, respectively. The analytical sensitivity of the DPX test determined by the German Red Cross Blood Donor Service was 0·6 and 3·8 IU/ml for HAV and B19. These multiplex and multi-dye blood screening assays represent a flexible NAT screening system for mini-pools between 6 and 96 samples per pool and fulfil all requirements of the European Pharmacopoeia for HCV and B19V testing of plasma for fractionation. The inclusion of a new multi-dye technology means discriminatory assays are no longer required for either test thus improving workflow, turn-around time and minimize the risk of obtaining a reactive result for which the virus cannot be identified.

High false‐negative rate of anti‐HCV among Egyptian patients on regular hemodialysis

Routine serological testing for hepatitis C virus (HCV) infection among hemodialysis (HD) patients is currently recommended. A dilemma existed on the value of serology because some investigators reported a high rate of false-negative serologic testing. In this study, we aimed to detect the false-negative rate of anti-HCV among Egyptian HD patients. Seventy-eight HD patients, negative for anti-HCV, anti-HIV, and hepatitis B surface antigen, were tested for HCV RNA by reverse transcriptase polymerase chain reaction (RT-PCR). In the next step, the viral load was quantified by real-time PCR in RT-PCR-positive patients. Risk factors for HCV infection, as well as clinical and biochemical indicators of liver disease, were compared between false-negative and true-negative anti-HCV HD patients. The frequency of false-negative anti-HCV was 17.9%. Frequency of blood transfusion, duration of HD, dialysis at multiple centers, and diabetes mellitus were not identified as risk factors for HCV infection. The frequency of false-negative results had a linear relation to the prevalence of HCV infection in the HD units. Timely identification of HCV within dialysis units is needed in order to lower the risk of HCV spread within the HD units. The high false-negative rate of anti-HCV among HD patients in our study justifies testing of a large scale of patients for precious assessment of effectiveness of nucleic acid amplification technology testing in screening HD patient.

The implementation of nucleic acid amplification technology testing for living tissue donors

There is a significant requirement within the United Kingdom for tissue grafts from living donors. To ensure safety, blood samples from these donors are tested for pathogens at donation, and at 180 days post donation. Nucleic acid amplification technology (NAT) permits more sensitive detection of pathogens in blood samples than serum antigen testing. NAT testing can be applied to samples from living tissue donors to eliminate the need to re-test these donors 180 days post-donation before grafts can be implanted. This has major financial and operational advantages for a tissue bank, and this manuscript describes how NAT testing was assessed and implemented by NHSBT Tissue Services. When compared to traditional serum antigen testing, NAT testing was more cost effective, more convenient for donors and resulted in a greater proportion of donated grafts being made available for transplant.

Quarantine Plasma: Quo vadis?

Upon the introduction of mandatory nucleic acid amplification technology (NAT) testing in Germany for HCV, quarantining of fresh frozen plasma (FFP) was reduced in 2002 from 6 to 4 months. In 2004 HIV-1 NAT and in 2005 anti-HBc testing were introduced to further reduce the residual transmission risks for transfusion relevant viruses. After testing more than 40 million donations by HCV NAT it became obvious that NAT testing has a very significant impact on viral blood safety by reducing the residual risk by a factor of 10. Only one documented HCV transmission occurred during more than 10 years of NAT testing in Germany, indicating that the remaining risk is marginal. Similar data were obtained for HIV-1. The question arises whether we could discontinue quarantining of FFP or further reduce the quarantining interval for retesting of the donor. This could facilitate logistics and reduce losses as quarantine FFP can be released earlier after donation and at regular donation intervals. Essential parameters for estimating the remaining infectious risks are the minimal infectious dose and replication kinetics of the viruses involved, the detection limits of the NAT tests applied, and the volume of plasma transfused. In essence it can be assumed that discontinuation of quarantining would only marginally increase the residual risk and that the reduction of the quarantine period to only 4 weeks would add an additional benefit to the viral safety of quarantine FFP.

Balance module allows consistent monitoring and documentation of the pooling process for nucleic acid amplification technology testing

When performing minipool testing of blood donations for transfusion-transmissible viruses, it is crucial to correctly dispense plasma from each donation into the pools. However, concerns regarding the monitoring and documentation of the pooling process exist. A balance module with a tube holder has been developed, which can be easily integrated into liquid handling platforms. The existing software monitors and evaluates every single dispensing from primary samples to the minipool to confirm liquid arrival. The weighing accuracy of the balance module is approximately 2 mg per dispensing episode. Ten thousand one hundred and fifty-six blood donations were tested on a Tecan Genesis RSP pipetting system. Thirty-one donations were not pipetted because the pipetting workstation identified a clot or sample shortage in the primary tube. The balance module exclusively detected another 18 mispipettings, which were outside of the acceptance criteria of 100 +/- 10%. The mean pipetted volume for these samples was 34.2 microl (range 0-87 microl). Visual inspection of the corresponding primary tubes showed blood clots, short sample or no apparent cause. The average deceleration of the pooling process, using the balance, was determined to be about 22%. With the novel liquid arrival check system, complete and consistent process documentation of pooling for nucleic acid amplification technology (NAT) testing is feasible. It enables blood banks to monitor and compare every single dispense made with predefined, required volumes. Results can be transferred to the laboratory information management system for automated selective exclusion of inaccurately dispensed samples. ONE SENTENCE SUMMARY: Monitoring and documenting the pooling process for NAT testing using a balance-based liquid arrival check system.

69. LABORATORY CONTRIBUTION FOR CONTROL OF CHLAMYDIA

Infection with Chlamydia trachomatis may present a wide spectrum of clinical symptoms in patients. Diagnosis based on presenting symptoms however may be of limited value in asymptomatic patients. Therefore, laboratory tests are utilized; in particular nucleic acid amplification technology (NAAT) based assays. In the recent years, NAAT testing has improved diagnosis of chlamydia to a great degree. The increased sensitivity of NAAT assays has also allowed for better detection of chlamydia in extragenital samples, diagnosis of lymphogranuloma venereum and has made the use of non-invasive self-collected samples possible. Chlamydia genotyping and sequence based comparisons are also increasingly being used in investigation of types present in the population as well as providing possibility of differentiating a new infection from re-infection. Such methods continue to play a major role not only in patient diagnosis but also in epidemiology and public health.

SI19 NAT or Not to NAT – This is the Dilemma?

The National Blood Service has undertaken a strategic review of Nucleic acid Amplification Technology (NAT) Testing used to screen blood for Hepatitis C Virus (HCV) at the request of the Department of Health.Three strategic options have been considered and are presented within this review:Continue testing (which in practice also includes developing this technology further as new automated platforms and multiplex tests become available).Substitute HCV antigen testing for NAT testingStop NAT HCV testingIn essence, this review concludes that NAT HCV testing is an extremely expensive intervention. Now that NAT testing is well developed by Blood Services world‐wide and has been in use to screen the UK population for some time, the consequences of pursuing each of the options presented can be predicted with accuracy.

Hepatitis B virus vaccination of blood donors – what costs may be expected?

Although the risk of transfusion-transmitted hepatitis B virus (TT-HBV) infection is very low, it still exists. Therefore, introduction of further precautions to reduce this risk is discussed at present. However, so far no data are available about the HBV vaccination status among blood donors (BDs). We compared HBV vaccination status of apheresis donors (ADs) of a university based and whole BDs (WBDs) of a Red Cross blood donation service using a standardized questionnaire. On the basis of these data, the estimated costs over 10 years for HBV vaccination were calculated for two different strategies and compared with the costs for HBV nucleic acid amplification technology (NAT) testing. 22.3% of the WBDs and 41.2% of the ADs indicated having received at least one HBV vaccine dose. This difference was related to the different demographic structures of the two BD populations (BDPs). With regard to the primary costs for the blood donation service, HBV vaccination of BDs could be an alternative to HBV NAT testing, especially for BDPs with an already high HBV vaccination rate and a high donation frequency.

Impact of nucleic acid amplification technology (NAT) in Italy in the three years following implementation (2001-2003).

The use of NAT technology to screen blood donations in Italy became mandatory on 28 June 2002, but had been available experimentally since 2001. During the transition period, an EIA test to detect hepatitis C core antigen (HCVcoreAg) had also been permitted. Considering the large number of blood transfusion centres in Italy, an initial reorganisation of the biological validation of blood units was necessary, with a partial centralisation of NAT testing. The Gruppo Italiano per lo Studio delle Malattie Trasmissibili con la Trasfusione (Italian Group for the Study of Transfusion-Transmissible Diseases) conducted a national survey evaluating NAT testing, based on an annual collection of data through a questionnaire sent to all centres. In the first three years of the investigation, 219 blood transfusion centres returned the questionnaires. In the period between January 2001 and December 2003, 3 894 894 blood donations were investigated for HCV RNA and 2 186 468 for HIV RNA. Of these, 12 were found to be HCV RNA positive and four HIV RNA positive, with an observed NAT versus antibody-based assay yield of 3.1/106 donations for HCV and 1.8/106 donations for HIV, respectively. Five of the 12 HCV RNA positive and anti-HCV negative donors had abnormal ALT values and their donations would have been discarded even in absence of NAT testing. Thus the final NAT yield for HCV is 1.79/106. The residual risk for HCV or HIV transmission by blood transfusion after NAT implementation is currently estimated to be extremely low in Italy.

Collaborative study to calibrate hepatitis C virus genotypes 2–6 against the HCV International Standard, 96/790 (genotype 1)

A major requirement of a hepatitis C virus (HCV) RNA nucleic acid amplification technology (NAT) assay validation is the ability of the assay to detect the six major genotypes of HCV with equivalent sensitivities. The aim of this study was to characterize and calibrate an HCV genotype panel for use in such studies. Panels consisting of the first International Standard (IS) for HCV RNA NAT assays (96/790; HCV genotype 1a) and isolates of genotypes 2-6 were sent to 17 laboratories worldwide which use a variety of NAT tests, both qualitative and quantitative. The HCV RNA content of each panel member was determined and the mean titre calculated in International Units/ml (IU/ml). The calculated mean titres (calibrated against the HCV International Standard), in log10 IU/ml, of the genotype 2-6 samples were 3.99, 3.81, 4.14, 4.18 and 4.61, respectively. An HCV genotype panel, calibrated in IU/ml, has been established and should be valuable for assay validation. All the genotypes were detected by all the assays used, but it was not possible to demonstrate that the genotypes were detected with equal efficiencies.

Blood screening by nucleic acid amplification technology: current issues, future challenges.

Nucleic acid amplification technology (NAT) is presently being evaluated in US clinical trials to determine the safety and efficacy of mini-pool testing for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA in the blood-donor population. Although the risk for transfusion-transmitted HIV and HCV infection is extremely low, there is still a small chance that blood donated by infected individuals before seroconversion can escape detection by current antibody-based assays. This report describes the amplification technologies being used and reviews several issues surrounding NAT-based blood screening. The performance features of NAT and current enzyme immunoassay technologies are compared, and the benefits of NAT in reducing transfusion-transmitted infections are discussed. The current US clinical trials of mini-pool NAT testing for HIV and HCV RNA have successfully identified preseroconversion infectious blood units. Although the current NAT-based screening systems are semiautomated, mini-pool testing represents an unprecedented innovation among government and nongovernment agencies in the highly regulated blood transfusion industry. Despite cost-effectiveness issues, based on the public perception of infectious diseases acquired through blood transfusion, NAT-based screening of the blood supply is expected to become a standard in transfusion medicine.