Search

The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses.

Multiple nucleic acid amplification tests (NATs) are available for the detection of SARS-CoV-2 in clinical specimens, including Laboratory Developed Tests (LDT), commercial high-throughput assays and point-of-care tests. Some assays were just recently released and there is limited data on their clinical performance. We compared the Xpert® Xpress SARS-CoV-2 (Cepheid) and Vivalytic VRI Panel (Schnelltest COVID-19) (Bosch) point-of-care tests with four high-throughput assays and one LDT, the cobas® SARS-CoV-2 test (Roche), the AllplexTM 2019-nCoV Assay (Seegene), the SARS-CoV-2 AMP (Abbott) Kit, the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona) as well as an assay using a SARS-CoV-2 RdRP gene specific primer and probe set. Samples from patients with confirmed SARS-CoV-2 infection, samples from the first and second SARS-CoV-2-PCR External Quality Assessment (EQA) (INSTAND e.V.) and a 10-fold serial dilution of a SARS-CoV-2 cell culture (SARS-CoV-2 Frankfurt 1) supernatant were examined. We determined that the NAT assays examined had a high specificity. Assays using the N gene as target demonstrated the highest sensitivity in the serial dilution panel, while all examined NAT assays showed a comparable sensitivity when testing clinical and EQA samples.

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at −20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

Isothermal nucleic acid amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are suitable for the development of a rapid, low-cost NAT that can be used at the POC. For demonstration of utility for global use, studies are needed to validate the performance of RT-LAMP for the detection of divergent subtypes. In this study, we designed and evaluated multiplexed HIV-1 integrase RT-LAMP primers to detect subtypes within group M, along with an RNase P positive internal processing and amplification control. Using a panel of 26 viral isolates representing the major circulating subtypes, we demonstrated detection of all isolates of subtypes A1, C, D, F1, F2, G, CRF01_AE, CRF02_AG, and two unique recombinant forms (URFs). A whole blood panel created with one representative isolate of each subtype was successfully amplified with the group M HIV-1 integrase and RNase P internal control primers. The group M HIV-1 RT-LAMP assay was further evaluated on 61 plasma specimens obtained from persons from Cameroon and Uganda. The sequence-conserved group M HIV-1 RT-LAMP primers, coupled to a low-cost amplification device, may improve diagnosis of acute infection at the POC and provide timely confirmation of HIV status.

Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37 °C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11 IU/ml (on board) and 7.21 IU/ml (off board) for HCV and 55.11 IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms.

Twenty-eight laboratories from 16 countries participated in a collaborative study to evaluate an HIV-1 RNA Genotype Reference Panel for use with nucleic acid-based tests (NAT). The Reference Panel consisted of 11 coded samples representing different HIV-1 genotypes (subtypes A-D, AE, F, G, AA-GH, groups N and O) as well as a negative diluent control. Each laboratory assayed the eleven panel members concurrently with the 1st International Standard for HIV-1 RNA (NIBSC Code 97/656) on at least three separate occasions and the data collated and analysed at NIBSC. Twenty-nine sets of data from NAT were received, 19 from quantitative and 10 from qualitative assays, with six different commercial assays and five "in-house" assays represented. The results showed that viruses from subtypes A-D and recombinant virus AE [CRF01_AE] were detected consistently, but that some assays had difficulty with the detection and quantification of viruses from subtypes F and G, a mixed recombinant virus AA-GH and a representative of group N. Furthermore, most assays failed to detect the group O representative. The study illustrated the limitations of some molecular assays particularly in detection of certain non-B genotypes which are important viruses in the global AIDS pandemic and illustrated the value of a well-characterised genotype panel. The panel has been established by the World Health Organisation's Expert Committee on Biological Standardisation as the 1st International Reference Panel HIV-1 RNA Genotypes (code 01/466).

The performance of twelve HIV combined p24 antigen and antibody assays available in Europe were compared. The assays were examined with a total of 1983 samples that included 1005 unselected HIV negative samples, 7 HIV-1 p24 Ag reference samples with HIV-1 Ag, 10 samples of a HIV antigen sensitivity commercial panel, 124 samples of 31 p24 antigen panels of different HIV-1 subtypes, 168 members of 24 HIV-1 seroconversion panels, 559 HIV-1 (groups M and O) antibody positive samples and 110 HIV-2 antibody positive samples. The specificity ranged from 99.4 to 100%. Ten of the 12 assays detected all anti-HIV positive samples irrespective of genotype while two assays missed one sample each (one subtype F and one subtype C). The combined assays could be classified into three groups. The first includes two assays (Enzygnost HIV Integral and Vironostika Ag/Ab) that have a clinical sensitivity similar to the two antibody only assays. The second includes the seven assays that detected infection after the p24 antigen only assay and show a delay from 3.3 to 5.17 days after HIV-1 RNA. The third group detected the infection before the p24 antigen assay and less than 3 days after nucleic acid testing (NAT). The improved ability to detect p24 Ag, at levels similar to specific HIV Ag assays, suggests that these new HIV combined Ag/Ab assays could replace p24 antigen only assays in situations for blood or organ screening when NAT is not feasible or not affordable.

Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naive chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P

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 World Health Organization (WHO) International Standard (IS) for human immunodeficiency virus type 1 (HIV-1) RNA is only available in limited amounts. It is critical to use the most common HIV-1 genotype as source for secondary standards, e.g. a National Standard for Taiwan. The objective of this study was to establish the first National Standard for HIV-1 RNA NAT assays in Taiwan. A collaborative study, including eleven laboratories from five different countries, was carried out to establish the HIV-1 RNA National Standard by calibration, in International Units (IU), against the WHO HIV-1 RNA IS. The HIV-1 RNA content for the candidate was quantitated by each laboratory in three independent assays and the results were collected and analyzed statistically. Overall, a high level of agreement among results was achieved from different laboratories. In addition, the stability study indicated that the candidate was stable for 24 months at −80 ± 5 °C. In conclusion, the candidate standard was established as the first National Standard for HIV-1 RNA for use in NAT assays in Taiwan. The standard is intended to be used for the quality control of HIV-1 NAT assays and as a quantitative reference material for HIV-1 NAT assays.

Nucleic acid testing (NAT) is valuable for screening blood donors for occult hepatitis B virus (HBV) infection and infection during the window period in countries where HBV is endemic, such as China. An “in-house” NAT (Triplex NAT) was developed for screening for HBV DNA, hepatitis C virus (HCV) RNA, and the human immunodeficiency virus type 1 (HIV-1) RNA. Using the Triplex NAT, a head-to-head comparative clinical evaluation was carried out against the most common commercial NAT used for blood screening in China: the Procleix Tigris System. A total of 33,025 specimens which were negative for Hepatitis B surface antigen, HCV antibody and HIV-1 antibody/antigen from potential blood donors were tested for HBV DNA, HCV RNA, and HIV-1 RNA by both the in-house Triplex assay and the commercially available Procleix Tigris System. Eleven specimens were detected as HBV positive by both NATs. Twelve specimens were detected as HBV positive by the Procleix Ultrio assay and the discriminatory assays, and not the Triplex. Twenty-eight specimens were detected as HBV positive by the Triplex and not the Procleix Ultrio. This study, combined with other data obtained in China, suggest that at least 50% HBV surface antigen negative but DNA-positive blood donations would be undetected using the current commercial NATs because of their insufficient sensitivity and/or Mini-Pool formatting strategies.

Nucleic acid amplification techniques have become the mainstay for ultimate sensitivity for detecting low levels of virus, including human immunodeficiency virus (HIV). As a sophisticated technology with relative expensive reagents and instrumentation, adoption of nucleic acid testing (NAT) can be cost inhibited in settings in which access to extreme sensitivity could be clinically advantageous for detection of acute infection. A simple low cost digital immunoassay was developed for the p24 capsid protein of HIV based on trapping enzyme-labeled immunocomplexes in high-density arrays of femtoliter microwells and constraining the diffusion of the enzyme–substrate reaction. The digital immunoassay was evaluated for analytical sensitivity for HIV capsid protein p24, and compared with commercially available NAT methods and immunoassays for p24, including 4th-generation antibody/antigen combo assays, for early detection of HIV in infected individuals. The digital immunoassay was found to exhibit 2000–3000-fold greater analytical sensitivity than conventional immunoassays reactive for p24, and comparable sensitivity to NAT methods. Assaying serial samples from 10 HIV-infected individuals, the digital immunoassay detected acute HIV infection as early as NAT methods, and 7–10 days earlier than conventional immunoassays. Comparison of assay results between the digital immunoassay and a quantitative NAT method from HIV infected serum exhibited a linear correlation R2>0.99. The data indicate that by constraining diffusion of the signal generation step of a simple sandwich immunoassay and enabling the digital counting of immunocomplexes, dramatic improvements in sensitivity to virus can be obtained to match the sensitivity of NAT at a fraction of the cost.

An international standard for the quantitation of HIV-2 RNA in plasma samples was developed. A collaborative study involving 29 laboratories from 15 countries was carried out in order to evaluate HIV-2 RNA candidate materials for use with nucleic acid-based tests (NATs). Candidate reference standards consisted of duplicate copies of two HIV-2 genotype A viruses, HIV-2 CAM2 and HIV-2 ROD and were coded S1–S4. Each laboratory assayed all four candidates on at least three separate occasions and data were collated and analysed at NIBSC. Of the data sets returned the majority were from qualitative assays. All assays detected both candidate standards with the exception of one commercial assay, the Nuclisens Easy Q, which was designed primarily for HIV-1 detection which did not detect HIV-2 CAM2 but showed good detection of HIV-2 ROD. This highlighted possible cross reactivity with HIV-2 ROD with some NAT primer/probe combinations; as a result the HIV-2 CAM2 material was established as the 1st international standard for HIV-2 RNA with an assigned unitage of 1000 International Units (IU) per ampoule and is available upon request from the National Institute for Biological Standardisation and Control (NIBSC) ( www.nibsc.ac.uk ).

Multiple nucleic acid-based techniques (NAT) have been implemented for testing blood and plasma donors for HIV-1 RNA which may be detected at an earlier stage of infection when HIV antigen or antibody is absent or below the limit of detection of current assays. The available NAT assays are based on different technologies. In order to evaluate the performance of nucleic acid-based techniques (NAT assays) and to allow accurate comparisons of results from different assays, it is essential to have well characterized specimens with known copy numbers as a standard. For this purpose, a comprehensive study was conducted to develop two HIV-1 RNA reference panels. The first (Panel 1) was prepared using a single specimen from the HIV-1 group M subtype B and consists of panel members with a wide range of HIV-1 RNA copy numbers. Panel 2 consists of 26 members representing HIV-1 group M subtypes A, C, D, E, F, G and groups O and N. For accurate determination of HIV-1 RNA copy numbers of each member of Panel 2, they were analyzed using various testing platforms/technologies available through the cooperation of five independent laboratories participating in the study. A consensus value for HIV RNA copy number was assigned to each member of Panel 2 based on statistical analysis of the data provided by the participants. Both panels could serve as reference panels to be used by manufacturers of HIV NAT tests to evaluate the sensitivity limits of their assays.

Many laboratories use working reagents/run controls to monitor the performance of their nucleic acid amplification techniques (NAT) for the measurement of HIV-1 RNA. A collaborative study was carried out in order to calibrate seven internationally available working reagents, QC105 (National Serology Reference Laboratory [NRL], Australia), B5 and B10 (Center for Biological Evaluation and Research [CBER], USA), Pelispy (Central Laboratory of the Netherlands Blood Transfusion Service [CLB], The Netherlands), PWS-1 and PWS-3 (National Institute for Biological Standards and Control [NIBSC], UK) and IRC (Virology Networks [VN], The Netherlands) against the 1st International Standard for HIV-1 RNA (code 97/656). Twenty-one laboratories from 12 different countries participated in the collaborative study and from the results it was determined that QC105 contained 4.0 log(10) International Units (IU)/ml, B5 2.2 log(10) IU/ml, B10 3.8 log(10) IU/ml, Pelispy 4.4 log(10) IU/ml, PWS-1 3.6 log(10) IU/ml, PWS-3 2.7 log(10) IU/ml and IRC 4.3 log(10) IU/ml. The seven working reagents calibrated in this international study may be used to validate and standardise the large number of qualitative and quantitative, commercial and in-house NAT assays that are currently being applied in the fields of blood safety and patient management. They will also help laboratories to comply with the sensitivity requirements that may be brought in by the regulatory authorities and may contribute to further harmonisation of guidelines on NAT published by organisations such as the European Medicines Evaluation Agency (EMEA), Paul-Ehrlich Institute and CBER, FDA.

Twenty-six laboratories from 10 different countries participated in a collaborative study to establish the 1st International Standard for HIV-1 RNA for use in nucleic acid-based techniques (NAT). Three candidate preparations were tested all based on genotype B viruses. The candidates were tested by each laboratory at a range of dilutions in four independent assays and the results collated and analysed statistically. All three candidates gave results that were tightly grouped, with little difference between the results from different laboratories or from the use of different assays. Studies of relative potency showed good agreement between laboratories. There were no significant differences between five commercial assay types, except that candidate XX showed a slightly lower potency compared to YY and ZZ with a single commercial assay. The reason for this was not established. Degradation studies showed that the freeze-dried preparations were stable at -20,4 and 20 degrees C for 26 weeks, the longest period studied, but that they became difficult to reconstitute after 3 weeks at 45 degrees C and 9 weeks at 37 degrees C. As a result of the study, the World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) established the preparation referred to as candidate YY (NIBSC Code No. 97/656) as the 1st International Standard for HIV-1 RNA for use with NAT with an assigned potency of 100000 International Units per vial.