Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.     

NAT: Perspectives for Cellular Components

The introduction of routine testing to detect viral genomes in donated blood was originally driven by requirements for plasma fractionation in relation to exclusion of hepatitis C virus (HCV) RNA. Nevertheless, it was obvious from the outset that a dual standard for fractionated products and individual blood components would be untenable. In many countries therefore, planning for introduction of nucleic acid testing (NAT) of blood incorporated progression to release of HCV RNA tested components. HCV was singled out because of its long seronegative 'window period', relatively high prevalence and incidence in blood donors, rapid burst time and high genome copy number during seroconversion. The latter properties made HCV particularly suitable for detection in pools of samples. If HCV RNA testing is required for release of labile components such as platelets, rapid provision of NAT results is vital because of short shelf life of platelets and the problems of delays when resolving the infectious unit in a reactive pool. For NAT release of labile components smaller sample pool sizes allow faster resolution of RNA positive units. Smaller pools involve high test throughput, the likely need for more testing laboratories and ensuing increased costs. Single sample testing is the ultimate extrapolation of reducing sample pool size. With reduced pool sizes or single sample testing, the option of testing for other viruses (e.g. HIV or HBV) singly or in multiplex also arises. The cost-benefit and incremental yield of such strategies in the light of 'combo' assays for HIV Ag/Ab and the recently described HCV Ag assay will require careful and objective assessment, together with re-appraisal of anti-HBc screening for detection of HBV infected donors at the "tail-end" of carriage.     

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.     

Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT)

Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.     

Application of nucleic acid amplification in clinical microbiology

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.     

Establishment of laboratory quality control indicators for NAT on blood donors' samples

In order to establish and screen nucleic acid test (NAT) quality control indicators for blood donor samples, C_T value of each test item were detected by NAT, internal and external quality controls, the number of unsuccessful mixed samples, the split positive rate, the rate of detection efficiency, and the equipment failure rate were collected. The "instant method" is used to establish the Levey-Jennings quality control chart, the mixed test positive rate, the rate of unqualified sample. Possion distribution is used to establish the quality control chart, and it is easily to operate and monitor NAT effectively. The results displayed that quality control charts were established, including unqualified sample Possion distribution quality control chart, the rate of equipment failure quality control chart, the failure rate of reagent batch and inefficient quality control chart, test result positive of Possion distribution quality control chart, quality control charts of NAT positive Possion distribution probability, the rate of split positive quality control charts, quality control charts of unsuccessful mixed samples, quality control chart of reagents effective rate, internal quality control chart, correctness, and NAT qualitative quality control chart. The study established quality control indicators throughout the whole NAT process, which were able to effectively improve NAT quality control and the efficacy of laboratory management.     

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.     

Alternative Molecular Tests for Virological Diagnosis

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.     

Analysis and application of NAT in Dongguan blood center more than 10 years

This report reviewed the efficacy of nucleic acid testing (NAT), derived from assaying and measuring data, using a Cobass201 system at the Dongguan blood center from 2008 to 2017. During this period, four blood screening models, each reflecting procedure improvements designed to improve residual risk (RR) assessment were assessed. A total of 716 846 blood donors were screened, detecting 1 395 positive by the mixed pool test, which were finally modified to 900 positive cases, after final detection by the separation-single test: detecting 6 HIV cases, 4 HCV cases and 890 HBV cases with a total positive rate of 1.25%. The lowest result was obtained from the twice administered enzyme-linked immunoassay (ELISA) test,used in conjunction with the single Cobas MPX v2.0 model,with rates of: 1/7 405 for HBV, 1/346 020 for HCV and 1/473 934 for HIV, respectively. NAT positive rate is not affected by different screening models. NAT is a recent detection method which can be used to good effect with ELISA, and is a worth while procedure for promoting blood transfusion safety.     

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.     

Molecular diagnosis of medical viruses

The diagnosis of infectious diseases has been revolutionized by the development of molecular techniques, foremost with the applications of the polymerase chain reaction (PCR). The achievable high sensitivity and ease with which the method can be used to detect any known genetic sequence have led to its wide application in the life sciences. More recently, real-time PCR assays have provided additional major contributions, with the inclusion of an additional fluorescent probe detection system resulting in an increase in sensitivity over conventional PCR, the ability to confirm the amplification product and to quantitate the target concentration. Further, nucleotide sequence analysis of the amplification products has facilitated epidemiological studies of infectious disease outbreaks, and the monitoring of treatment outcomes for infections, in particular with viruses which mutate at high frequency. This review discusses the applications of qualitative and quantitative real-time PCR, nested PCR, multiplex PCR, nucleotide sequence analysis of amplified products and quality assurance with nucleic acid testing (NAT) in diagnostic laboratories.     

Challenges in the Testing of Non-Heart-Beating Cadavers for Viral Markers: Implications for the Safety of Tissue Donors

Natural changes that occur in blood and tissue after death may result in false positive results in antigen and antibody detection tests performed to identify markers of viral infection in potential tissue donors. Such tissue, which might otherwise be acceptable for therapeutic purposes, would not meet current standards for safe tissue banking. This is especially important in the context of insufficiency in the tissue supply. In this study, a series of blood samples collected during routine post-mortem examination was assayed using a range of commercially available kits for the detection of HBsAg, anti-HCV and anti-HIV 1 + 2 antibody/antigen. Results of tests on 104 samples collected from 97 individuals indicate that some kits result in a higher number of initial reactive samples than others. Approximately 40% of samples were reactive in one or more HBsAg assay, less than 10% in at least one anti-HIV kit and only 1 sample at low level on an anti-HCV kit. Liver or lymph node samples from individuals whose serum sample gave reactive results in antigen/antibody assays were tested for viral nucleic acid in the corresponding nucleic acid amplification test. Only one individual’s sample was confirmed to test positive for HBsAg in a confirmatory neutralisation test and by nucleic acid amplification technology, and a second individual whose serum was scored reactive for anti-HCV, but negative for HBsAg, had a liver sample which was HBV DNA positive and HCV RNA negative. The results of the study indicate that antibody/antigen assays are not as specific as NAT using state of the art DNA extraction techniques. Both types of assay complement each other and used together will help assure the safety of tissues for transplantation.     

食源性病毒核酸恒温检测技术研究进展

食源性病毒已成为全球引发食品安全事件的重要病原,对新型检测技术的不断发展提出了严峻的挑战.早期PCR技术在病原检测领域中的应用,推动了对食源性病毒的全面认识.近年来核酸恒温检测技术发展迅速,包括环介导等温扩增技术、重组酶聚合酶扩增技术、核酸序列依赖性扩增技术、链置换扩增技术、滚环扩增技术等,在抗复杂基质干扰、装备要求低...     

Safety Issues for Plasma Derivatives and Benefit from NAT Testing

Manufacturing processes for plasma derivatives are in general highly effective for removal or inactivation of enveloped viruses and the products are safe with regard to the clinically important viruses HIV, HCV and HBV. They are not so effective for the elimination for non-enveloped viruses, especially Parvovirus B19 (B19). A certain risk remains of B19 contamination for some plasma derivatives that is caused, firstly, by the occurrence of highly contaminated donations (up to 10(14)genomes/ml) and secondly, by the extreme heat resistance and small size of B19 which makes it difficult to remove or inactivate. NAT is a beneficial tool for detection of virus contamination. It is routinely used for the detection of HCV-RNA in plasma pools, thereby preventing the processing of HCV-RNA positive material. NAT assays may also be valuable for testing the removal of viruses during manufacturing. This may be especially important if a virus cannot be tested by infectivity assays.     

DNA detection using recombination proteins

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.