International Reference Panel Research Articles

A validated and standardized pseudotyped microneutralization assay as a safe and powerful tool to measure LASSA virus neutralising antibodies for vaccine development and comparison.

Over the past few decades, World Health Organization (WHO) has made massive efforts to promote the development of a vaccine against Lassa virus (LASV), one of the top ten priority pathogens for research and development under the WHO R&D Blueprint for Emerging Infections. To date, several vaccines are at different stages of development. In this scenario, a validated and standardised assay to measure LASV neutralising antibodies is urgently needed for vaccine development and comparison. The neutralisation assay remains the gold standard for determining antibody efficacy. Here we have proposed a safe and validated pseudotyped neutralisation assay for LASV, taking advantage of the development of the first WHO International Standard and Reference Panel for Anti-Lassa Fever (NIBSC code 21/332). The proposed results demonstrate that the pseudotyped luciferase neutralisation assay is a specific serological test for the measurement of LASV neutralising antibodies without cross-reacting with standard sera specific for heterologous viral infections. In addition, the assay is accurate, precise, and linear according to criteria and statistical analyses defined and accepted by international guidelines.

The characterization of genome sequences diversity of Pseudomonas aeruginosa strains from international reference panel using wide range of in silico techniques

Pseudomonas aeruginosa is an important pathogen in patients suffering from Cystic Fibrosis as well as acute opportunistic infections in people without it. For the reason of P. aeruginosa having a broad range of habitats it’s diversity and adaptability lead it to be a very diverse species. Previous attempts of classifying P. aeruginosa strains based on their biochemical and genetic characteristics were made. In presented studied we performed additional characteristic of P. aeruginosa panel strains genomes using wide range of in silico approaches, including Single Nucleotide Polymorphisms (SNPs) - based phylogeny, as well as pan-genome and Intergenic Regions (IGRs) investigation. We shed light on strains diversity, expanding our knowledge about the strains assembled in this international panel. The results of our study may become the basis for further research aimed at fully understanding the pathogenesis of P. aeruginosa.

A validate and standardized pseudotyped microneutralization assay as a safe and powerful tool to measure LASSA neutralising antibodies for vaccine development and comparison

Background Over the past few decades, WHO has made massive efforts to promote the development of a vaccine against Lassa virus (LASV), one of the top ten priority pathogens for research and development under the WHO R&D Blueprint for Emerging Infections. To date, several vaccines are at different stages of development. In this scenario, a validated and standardised assay to measure LSV neutralising antibodies is urgently needed for vaccine development and comparison. Methods The neutralisation assay remains the gold standard for determining antibody efficacy. Here we have proposed a safe and validated pseudotyped neutralisation assay for LASV, taking advantage of the development of the first WHO International Standard and Reference Panel for Anti-Lassa Fever (NIBSC code 21/332). Results and Conclusions The proposed results demonstrate that the pseudotyped luciferase neutralisation assay is a specific serological test for the measurement of LASV neutralising antibodies without cross-reacting with standard sera specific for heterologous viral infections. In addition, the assay is accurate, precise, and linear according to criteria and statistical analyses defined and accepted by international guidelines.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (<0.01%IS) based on the qPCR test results In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation

Administration of Bacteriophages via Nebulization during Mechanical Ventilation: In Vitro Study and Lung Deposition in Macaques.

Bacteriophages have been identified as a potential treatment option to treat lung infection in the context of antibiotic resistance. We performed a preclinical study to predict the efficacy of delivery of bacteriophages against Pseudomonas aeruginosa (PA) when administered via nebulization during mechanical ventilation (MV). We selected a mix of four anti-PA phages containing two Podoviridae and two Myoviridae, with a coverage of 87.8% (36/41) on an international PA reference panel. When administered via nebulization, a loss of 0.30-0.65 log of infective phage titers was measured. No difference between jet, ultrasonic and mesh nebulizers was observed in terms of loss of phage viability, but a higher output was measured with the mesh nebulizer. Interestingly, Myoviridae are significantly more sensitive to nebulization than Podoviridae since their long tail is much more prone to damage. Phage nebulization has been measured as compatible with humidified ventilation. Based on in vitro measurement, the lung deposition prediction of viable phage particles ranges from 6% to 26% of the phages loaded in the nebulizer. Further, 8% to 15% of lung deposition was measured by scintigraphy in three macaques. A phage dose of 1 × 109 PFU/mL nebulized by the mesh nebulizer during MV predicts an efficient dose in the lung against PA, comparable with the dose chosen to define the susceptibility of the strain.

Selection of a SARS-CoV-2 antibody quantification method and development of an antibody reference standard for ELISA to test immunoglobulin preparations

The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method; the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control.The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations.Materials and methods: the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay.Results: the authors validated an ELISA method for SARS-CoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG).Conclusions: the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.

Hepatitis E virus: Whole genome sequencing as a new tool for understanding HEV epidemiology and phenotypes.

Hepatitis E Virus (HEV) is emerging as a public health concern across Europe and tools for complete genome data to aid epidemiological and virulence analysis are needed. The high sequence heterogeneity observed amongst HEV genotypes has restricted most analyses to subgenomic regions using PCR-based methods, which can be unreliable due to poor primer homology. We designed a panel of custom-designed RNA probes complementary to all published HEV full genome NCBI sequences. A target enrichment protocol was performed according to the NimbleGen® standard protocol for Illumina® library preparation. Optimisation of this protocol was performed using 40 HEV RNA-positive serum samples and the World Health Organization International Reference Panel for Hepatitis E Virus RNA Genotypes for Nucleic Acid Amplification Technique (NAT)-Based Assays and related reference materials. Deep sequencing using this target enrichment protocol resulted in whole genome consensus sequences from samples with a viral load range of 1.25 × 104-1.17 × 107 IU/mL. Phylogenetic analysis of these sequences recapitulated and extended the partial genome results obtained from genotyping by Sanger sequencing (genotype 1, ten samples and genotype 3, 30 samples). The protocol is highly adaptable to automation and could be used to sequence full genomes of large sample numbers. A more comprehensive understanding of hepatitis E virus transmission, epidemiology and viral phenotype prediction supported by an efficient method of sequencing the whole viral genome will facilitate public health initiatives to reduce the prevalence and mitigate the harm of HEV infection in Europe.

PB2202 VALIDATION OF THE PRIMERDESIGN® QUANTITATIVE ALLELE SPECIFIC AMPLIFICATION KIT FOR THE DETECTION OF THE JAK2V617F MUTATION

Background:Janus Kinase 2 (JAK2) is part of a group of tyrosine kinases which are involved in cellular signalling. A point mutation in codon 617 of the JAK2 gene (JAK2 V617F) causes a ‘gain of function’ effect of this process, stimulating proliferation of the myeloid cell lines, including erythrocytes and platelets, as well as causing myelofibrosis in the bone marrow. Testing for this specific mutation using an allele‐specific PCR kit at our facility (NHLS‐TAD), Pretoria] should provide quick and reliable results to investigate suspected myeloproliferative neoplasms. As part of method comparison, 15 specimens known to be positive for the JAK2V617F mutation and 19 specimens known to be JAK2 negative, as previously assessed using PCR with melting curve analysis at Charlotte Maxeke Johannesburg Academic Hospital, will be analysed using the Primerdesign Quasa test kit on the BioRad CFX 96 platform. Results of the two PCR tests will be compared to evaluate accuracy of the kit.Aims:To validate the Primerdesign® quantitative allele specific amplification (Quasa) kit for detection of the JAK2V617F mutation in DNA extracted from whole blood samples.Methods:Patients’ samples, previously tested at the National Health Laboratory Service laboratory, Charlotte Maxeke Johannesburg Academic Hospital, will be used. These comprise both whole blood collected in EDTA as well as genomic DNA. 15 specimens known to be positive for the JAK2V617F mutation as well as 19 JAK2 negative specimens will be analysed as part of method comparison. We have chosen a sample size of 34 due to the substantial cost of each kit and we are limited due to financial constraints. The WHO international reference panel for genomic JAK2V617F with proposed percentages of 0, 0.03, 1, 10.8, 29.6, 89.5 and 100% will be used for quantitative analysis of the kit. In addition, we will be analysing a diluted JAK2V617F WHO sample to confirm the manufacturer's claim of an analytical sensitivity of 0.1%. Specimens already analysed will be re‐tested using the Primerdesign® quasa kit on the CFX96 real‐time PCR platform (Bio Rad).Results:There is a 95% concordance between the results obtained from the index test (Primerdesign kit) when compared to the test of reference (LightCycler). Precision includes repeatability and reproducibility, both of which were shown in this study. We were able to detect 0.1% mutant DNA in background wild type DNA as claimed by the manufacturer. Therefore the analytical sensitivity was at 0.1%. The ability of the test to not detect the mutation in a known negative sample was at 100% concordance. Therefore analytical specificity was 100%Summary/Conclusion:The Primerdesign allele specific kit has the capability of being able to produce qualitative as well as quantitative results based on the manufacturer's instructions. Being able to provide this diagnostic test in our department will decrease turn‐around time and improve patient care.

Development of a World Health Organization International Reference Panel for different genotypes of hepatitis E virus for nucleic acid amplification testing

BackgroundGlobally, hepatitis E virus (HEV) is a major cause of acute viral hepatitis. Epidemiology and clinical presentation of hepatitis E vary greatly by location and are affected by the HEV genotype. Nucleic acid amplification technique (NAT)-based assays are important for the detection of acute HEV infection as well for monitoring chronic cases of hepatitis E. ObjectivesThe aim of the study was to evaluate a panel of samples containing different genotypes of HEV for use in nucleic NAT-based assays. Study designThe panel of samples comprises eleven different members including HEV genotype 1a (2 strains), 1e, 2a, 3b, 3c, 3e, 3f, 4c, 4g as well as a human isolate related to rabbit HEV. Each laboratory assayed the panel members directly against the 1st World Health Organization (WHO) International Standard (IS) for HEV RNA (6329/10) which is based upon a genotype 3 a strain. ResultsThe samples for evaluation were distributed to 24 laboratories from 14 different countries and assayed on three separate days. Of these, 23 participating laboratories returned a total of 32 sets of data; 17 from quantitative assays and 15 from qualitative assays. The assays used consisted of a mixture of in-house developed and commercially available assays. The results showed that all samples were detected consistently by the majority of participants, although in some cases, some samples were detected less efficiently. ConclusionsBased on the results of the collaborative study the panel (code number 8578/13) was established as the “1st International Reference Panel (IRP) for all HEV genotypes for NAT-based assays” by the WHO Expert Committee on Biological Standardization. This IRP will be important for assay validation and ensuring adequate detection of different genotypes and clinically important sub-genotypes of HEV.

Detection and quantification of hepatitis E virus in the absence of IgG and IgM anti-HEV in HIV-positive patients.

To improve RT-qPCR with an internal control and a synthetic standard curve to detect HEV in HIV co-infected patients. A single-stranded RNA (ssRNA) and a double-stranded DNA (dsDNA) synthetic curve were designed, compared to the international reference panel for HEV genotypes, and tested to quantify and detect a reference panel for HEV genotypes. The detection limit of the RNA synthetic curve (50 copies per ml) was better than the DNA synthetic curve (100 copies per ml) and the WHO standard curve (250 copies per ml). Then, 280 serum samples from HIV-positive patients were tested for HEV RNA, which was detected in 3·6% of serum samples. The viral load ranged from 2×102 copies per ml to 4·78×108 copies per ml. HEV IgM/IgG antibodies were not detected in the RNA-positive patients. Sequencing analysis of HEV showed that the virus belongs to genotype 3 (HEV GT3). Real-time PCR was a useful tool to estimate co-infection with HEV/HIV, even in patients with low viral loads and undetectable anti-HEV IgG and IgM antibodies. Hepatitis E virus genotype 3 (HEV GT3) has been associated with silent chronic hepatitis and cirrhosis in HIV-positive subjects worldwide, but there is a lack of data on this co-infection in Brazil.

Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Hepatitis E Virus (HEV) Detection and Quantification by a Real-Time Reverse Transcription-PCR Assay Calibrated to the World Health Organization Standard for HEV RNA

Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.

Development of Xpert® BCR-ABL Ultra, an Automated and Standardized Multiplex Assay with Required Performance Characteristics for BCR-ABL1 Quantitative Measurement on an International Reporting Scale

Monitoring BCR-ABL transcript levels in patients on TKI therapy using real-time quantitative PCR is standard of care in the management of CML, and achieving sensitivity to 4.5 logs below baseline is important in patients being considered for TKI discontinuation. In order to accurately assess treatment response, it is essential to ensure that variability in testing methodologies is tightly controlled. This has resulted in substantial efforts to standardize BCR-ABL molecular measurement across laboratories by introducing an International Scale (IS). Here we describe the development and analytical evaluation of Xpert® BCR-ABL Ultra, an automated cartridge-based BCR-ABL monitoring assay incorporating lot-specific standardization to the International Scale as defined by WHO.The BCR-ABL Ultra cartridge simplifies the generation of highly accurate and reproducible assay results by integrating nucleic acid extraction, target amplification, and quantification of the BCR-ABL b3a2 or b2a2 transcripts relative to ABL in peripheral blood specimens. The assay turn-around-time is approximately 2.5 hours and has a %BCR-ABL/ABL(IS) dynamic range of ~6 logs, including two key milestones for the assessment of clinical response at ~10% (IS) (SD=~0.08Log) and ~0.0033% (IS) (SD=~0.25Log) (CMR4.5). Lot-to-lot standardization is achieved using a secondary standard panel of controls derived from and aligned to the first WHO international genetic reference panel. The secondary standards are produced at Cepheid as part of the assay kit manufacturing process and applied to every production lot of cartridges in order to improve assay reliability and reporting comparability. Analytical sensitivity studies using spiked-in CML cell lines demonstrated a limit of detection (LoD) and limit of quantification (LoQ) of 0.003% BCR-ABL/ABL (IS) with a limit of blank (LoB) of 0.00017%. In an analytical specificity study, assay results were negative in 100% of 70 known CML-negative samples, including 20 AML or ALL patients and 50 healthy donors. A study was further conducted to estimate the lower limit of clinical sensitivity using blood from 12 CML patients on TKI therapy who had achieved and maintained MMR with reporting below 0.05% (IS). Eleven out of twelve low CML subjects were detected in at least 19 out of 20 replicates tested per subject over a range of 0.038% (SD=~0.17Log) to 0.0011% (IS) (SD=~0.4Log).In conclusion, the Xpert BCR-ABL Ultra assay demonstrates sensitivity in the range necessary to make clinical decisions regarding the continuation of TKI therapy in patients achieving CMR4.5, and every production lot of cartridges is assigned a unique conversion factor guaranteeing alignment to the WHO International Scale. DisclosuresUy:Novartis: Research Funding.

Performance of two commercially available BCR-ABL1 quantification assays that use an international reporting scale.

Quantifying the BCR-ABL1 rearrangement is important for monitoring chronic myelogenous leukemia (CML). To standardize BCR-ABL1 quantification, the World Health Organization (WHO) established the first international genetic reference panel. Here, we compared the BCR-ABL1 levels determined using international scale (IS)-based commercially available assays. BCR-ABL1 transcripts were quantified using two IS-based assays. 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 dilutions of the b3a2 positive RNA were used for evaluating linearity, precision, and limit of detection. Correlation of the assay was evaluated by using DNA obtained from CML patients carrying the BCR-ABL1 b3a2 and b2a2 types. Both Ipsogen and Asuragen assays showed fine linearity with reasonable %CV. LOD of each assay was calculated as 0.003% for Ipsogen, and 0.005% for Asuragen. By comparing the results that were lower than 10% by either one of the assay, Ipsogen and Asuragen results showed an overall good linear correlation with a tendency for the Ipsogen assay to show slightly higher levels than the Asuragen assay for b3a2 transcript. For b2a2, the tendency was opposite, with Asuragen showing higher values than the Ipsogen. Two commercially available IS-based BCR-ABL1 assays showed an overall good quantitative correlation. It should be taken into consideration that each assay tended to produce higher values than the other, depending on the BCR-ABL1 subtypes, suggesting that a separate conversion factor for each subtype can be more helpful when BCR-ABL1 transcript levels are converted into IS.

A new quantitative PCR for human parvovirus B19 genotypes

Parvovirus B19 (B19V) is a minute ssDNA virus associated with a wide range of diseases from childhood erythema to fetal death. After primary infection, the viral genomes persist lifelong in solid tissues of most types. Quantification of the viral DNA is important in the timing of primary infection, assessment of tissue persistence and screening of blood donor plasma. In this study, we present a new PCR assay for detection and quantification as well as for differentiation of all three B19V genotypes.A new B19V qPCR was designed to target a 154-bp region of the NS1 area. Serum, plasma and solid tissue samples were suitable for testing in the assay. The WHO International Reference Panel for Parvovirus B19 Genotypes was utilized to validate the assay for detection of different genotypes of B19V in clinical material. Each panel member yielded, by the new qPCR, a quantity similar to the one reported by National Institute for Biological Standards and Control (NIBSC). The qPCR was specific for B19V and amplified and quantified all three genotypes with detection sensitivities of ≤10 copies/reaction. The differentiation of B19V genotypes was performed by Sanger sequencing of the amplified products.

A Single-tube real-time RT-PCR assay for RNA detection and quantification of genetically diverse HIV including rare HIV-1 non-m groups and HIV-2

The RT-PCR method with real-time fluorescence detection was used for the development of a prototype of a diagnostic kit for reliable diagnosis of genetic variants of RNA of HIV-1 groups M, N, O, and P and RNA of HIV-2 in blood plasma and serum. The kit is tolerant to nucleotide mismatches, provides a broad linear range of concentrations of HIV RNA, 100% analytical specificity, and adequate analytical sensitivity: 42 ME/mL (HIV-1 group M), 45 copies/mL (HIV-2), 92 copies/mL (HIV-1 group O), and 90 copies/mL (HIV-1 group N). The kit provided successful detection and measurement of HIV RNA concentration in all the samples of the international reference panel for HIV-1 genotypes. The results of the test correlate with the results of commercial tests.

S101 Pathogen associated molecular patterns in cystic fibrosis pathogens: Analysis of peptidoglycan structure

BackgroundPathogen associated molecular patterns (PAMPS) are key virulence determinants in triggering immune mediated damage in cystic fibrosis. The lipopolysaccharide (LPS) in Pseudomonas aeruginosa strains isolated from CF-lungs has “CF specific”...

Keeping Pace with Parvovirus B19 Genetic Variability: a Multiplex Genotype-Specific Quantitative PCR Assay

Three genotypes have been identified within the parvovirus B19 species (B19V), and such genetic diversity may have significant implications for the development of molecular detection assays. In the present study, B19V genetic variability has been examined on a subset of genomic sequences available in the NCBI nucleotide database, and a quantitative PCR (qPCR) assay able to detect, differentiate, and quantify all viral variants has been established. The designed primers and probes have been used for the development of alternative detection formats, based on a combined use of intercalating dye and genotype-specific hydrolysis probes. The qPCR assay analytical performances have been determined on the 1st WHO International Reference Panel for Parvovirus B19 Genotypes. The developed qPCR protocols allow for the detection of genotypes 1 to 3 with equal accuracy, and with a limit of detection (LOD) of 200 IU/ml. A comparison of routine performance was carried out with respect to a previously established assay specifically validated on B19V genotype 1. For 130 clinical samples analyzed, 126 showed concordant results (31 positive and 97 negative), while 4 showed discordant results. Overall, the genotype-specific qPCR assay showed a sensitivity of 93.94% and a specificity of 97.94%, with an agreement rate of 96.92%. The proposed qPCR assay and the alternative protocols developed, each with robust performance, may allow choice with respect to operational systems and diagnostic requirements and might contribute to provide a more reliable diagnostic service and epidemiological surveillance of B19 virus.

Performance of hepatitis B surface antigen tests with the first WHO international hepatitis B virus genotype reference panel

BackgroundStandardization of hepatitis B surface antigen (HBsAg) tests is indispensable for consistent quality and comparability. Ideally, the assays should detect all known hepatitis B virus (HBV) genotypes equally well. ObjectiveDevelopment of an HBV genotype reference panel for HBsAg assays representing the most prevalent HBV subgenotypes to address commutability and traceability of the heat-inactivated 2nd WHO International Standard (IS) for HBsAg in relation to native HBsAg and to HBV genotypes. Study designAn HBV panel of 15 non-inactivated lyophilized specimens representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H was evaluated in parallel to the IS by 15 laboratories using 19 different HBsAg tests and tree unitages. The virus content of the samples was reduced by ultracentrifugation and dilution to <2×104IU HBV DNA/mL. ResultsTwenty-two qualitative and 6 quantitative data sets were evaluated. Overall, the results demonstrated consistent detection of HBV genotypes by the majority of tests with a mean potency variability relative to the IS of 36%. Some assays showed significant genotype-dependent differences in analytical sensitivity. Some tests were more sensitive with the IS, others less. On average, one IU HBsAg corresponded to 0.88±0.20ng HBsAg protein. ConclusionsThe panel was accepted by the WHO as the “1st International Reference Panel for HBV genotypes for HBsAg-based assays”. The panel is a helpful complementation to the IS to validate HBV genotype specific analytical test sensitivities.

Erratum: Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome

Erratum: Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome