Evaluation of the first automated thyroglobulin assay

The aim of this study was to investigate technical and analytical performance of the first automated thyroglobulin (Tg) assay (DPC-Immulite(R); Diagnostic Products Corporation, Los Angeles, USA). In imprecision studies using several human serum pools ranging from 21 to 58 replicates, a coefficient of variation of 9.0 % was obtained at a mean Tg concentration of 0.84 ng/ml and of 6.1 % at a Tg concentration of 62.1 ng/ml. In a method comparison with a non-automated assay (BRAHMS LUMItest Tg(R), BRAHMS, Berlin, Germany) using 383 sera of 303 patients with thyroid carcinoma, regression analysis according to Passing and Bablock yielded in the following equation: Immulite Tg=1.6 x BRAHMS Tg - 0.1 ng/ml (Pearson's r=0.979). Sera obtained from 59 patients with thyroid carcinoma enabled comparative follow-up studies; in all cases qualitative agreement was found with regard to increase or decrease of serum Tg; in eight cases, however, Tg was detected with the Immulite assay but not with the BRAHMS assay. Further follow-up proved the presence of thyroid tissue in these patients. From these and further methodological data (dilution linearity, interference studies, carry-over study, high-dose hook properties, and short report time) it is concluded that the DPC-Immulite Tg assay meets the requirements of routine diagnostic use

Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta)

CITATION: Clarke, C., et al. 2017. Development and evaluation of a diagnostic cytokine-release assay for mycobacterium suricattae infection in meerkats (Suricata suricatta). BMC Veterinary Research, 13:2, doi:10.1186/s12917-016-0927-x.The original publication is available at http://bmcvetres.biomedcentral.comBackground: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. Results: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. Conclusion: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.http://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0927-xPublisher's versio

Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay.

Background Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84–100), whiles the sensitivity was 88% (95% CI, 77–95). Conclusion The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure. Author summary Current diagnostic methods to detect M. ulcerans suffer from delayed time-to-results in most endemic countries by the prolonged period of time for the shipment and storage of samples to a distant, centralized laboratory. The M. ulcerans recombinase polymerase amplification assay (Mu-RPA) is a new, rapid diagnostic test developed for the detection of M. ulcerans infection, known commonly as Buruli ulcer, a chronic, debilitating, necrotizing disease of the skin and soft tissues. This assay is suitable for use on a portable detection device, with the potential to be used for quick diagnosis at the point of need, providing timely results to health workers at Buruli ulcer treatment clinics

Non-equivalence of anti-Müllerian hormone automated assays—clinical implications for use as a companion diagnostic for individualised gonadotrophin dosing

STUDY QUESTION Can anti-Müllerian hormone (AMH) automated immunoassays (Elecsys® and Access) be used interchangeably as a companion diagnostic for individualisation of follitropin delta dosing? SUMMARY ANSWER The Access assay gives systematically higher AMH values than the Elecsys® assay which results in over 29% of women being misclassified to a different follitropin delta dose. WHAT IS KNOWN ALREADY Follitropin delta is the first gonadotrophin to be licenced with a companion diagnostic, the Roche Elecsys® AMH Plus assay. Alternative automated AMH assays including the Beckman Coulter Access immunoassay are considered to provide similar results, but clarification of their suitability as an off-licence companion diagnostic for follitropin delta is required. STUDY DESIGN, SIZE, DURATION We systematically searched the existing literature for studies that had measured AMH using both automated assays in the same cohort of women. Individual paired patient data were acquired from each author and combined with unpublished data. PARTICIPANTS/MATERIALS, SETTING, METHODS We identified five eligible prospective published studies and one additional unpublished study. A 100% response from the authors was achieved. We collected paired AMH data on samples from 848 women. Passing–Bablok regression and Bland–Altman plots were used to compare the analytical performance of the two assays. The degree of misclassification to different treatment categories was estimated should the Access AMH be used as a companion diagnostic instead of the Elecsys AMH in determining the dosing of follitropin delta. MAIN RESULTS AND THE ROLE OF CHANCE The Passing–Bablok regression shows a linear relationship (Access = −0.05 + 1.10 × Elecsys). The Access assay systematically gave higher values by an average of 10% compared with the Elecsys assay (slope = 1.10, 95% CI: 1.09 to 1.12). The average of the difference between the two assays was 2.7 pmol/l. The 95% limits of agreement were −11.7 to 6.3. Overall 253 (29.3%) women would have received an inappropriate follitropin delta dose if the Beckman Coulter Access assay was used. Specifically, a substantial proportion of women (ranging from 49% to 90% depending on the AMH category) would receive a lower dose of follitropin delta based on the Access AMH assay. Up to 10% (ranging from 2.5% to 10%) of women with high ovarian reserve would have been misclassified to a greater dose of follitropin delta based on the Access AMH assay. LIMITATIONS REASONS FOR CAUTION We compared the values of the two principal automated assays, extrapolation of our findings to other automated AMH assays would require similar comprehensive examination. WIDER IMPLICATIONS OF THE FINDINGS An international standard for the calibration of the automated AMH assays is warranted to facilitate efficient use of AMH as a companion diagnostic. The variable calibration of alternative automated AMH assays may adversely impact on the performance of the follitropin delta dosing algorithm. STUDY FUNDING/COMPETING INTEREST(S) No formal funding has been received for this study. SI is funded by a UK Medical Research Council skills development fellowship (MR/N015177/1). SMN has received speakers fees, travel to meetings and participated in advisory Boards for Beckman Coulter, IBSA, Ferring Pharmaecuticals, Finox, Merck Serono, Merck and Roche Diagnostics. SMN has received research support from Ansh laboratories, Beckman Coulter, Ferring Pharmaceuticals and Roche Diagnostics

From aptamer-based biomarker discovery to diagnostic and clinical applications: an aptamer-based, streamlined multiplex proteomic assay

Recently, we reported an aptamer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

Standardization of sequencing coverage depth in NGS: Recommendation for detection of clonal and subclonal mutations in cancer diagnostics

The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30 mutated reads for a targeted NGS mutation analysis of >= 3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.Web of Science9art. no. 85

tmRNA - a novel high-copy-number RNA diagnostic target - its application for Staphylococcus aureus detection using real-time NASBA

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target

Validation of an automated enzyme immunoassay for interleukin-6 for routine clinical use

Serum levels of Interleukin-6 (IL-6), a proinflammatory cytokine, are increased in early stages of inflammatory diseases such as infection and sepsis. Assay systems which permit its measurement within a few hours and as a single measurement have not been reported so far. We therefore evaluated a now commercially available automated method for IL-6 measurement on the Cobas Core(R) immunological analyzer (Roche Diagnostic Systems) which enables single IL-6 measurement within about 1 hour. The automated assay correlates well with an established, manual microtiter plate assay (Biosource GmbH) which uses the same antibodies and reagents (r=0.98). Accuracy of the automated method was established by adding known amounts of IL-6 international reference preparation. Recovery of the international standard was in the range of 92-104%. The automated assay had a precision of singletons below 6% and was linear up to 2800 pg/ml. This automated assay provides a suitable, convenient and time saving method for measurement of IL-6 serum levels in the routine clinical laboratory

Accurate PCR detection of influenza A/B and respiratory syncytial viruses by use of Cepheid Xpert Flu+RSV Xpress Assay in point-of-care settings: Comparison to Prodesse ProFlu+

ABSTRACT The Xpert Flu+RSV Xpress Assay is a fast, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A and B viruses and respiratory syncytial virus (RSV) performed on the Cepheid GeneXpert Xpress System. The objective of this study was to establish performance characteristics of the Xpert Flu+RSV Xpress Assay compared to those of the Prodesse ProFlu+ real-time reverse transcription-PCR (RT-PCR) assay (ProFlu+) for the detection of influenza A and B viruses as well as RSV in a Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) setting. Overall, the assay, using fresh and frozen nasopharyngeal (NP) swabs, demonstrated high concordance with results of the ProFlu+ assay in the combined CW and non-CW settings with positive percent agreements (PPA) (100%, 100%, and 97.1%) and negative percent agreements (NPA) (95.2%, 99.5%, and 99.6%) for influenza A and B viruses and RSV, respectively. In conclusion, this multicenter study using the Cepheid Xpert Flu+RSV Xpress Assay demonstrated high sensitivities and specificities for influenza A and B viruses and RSV in ∼60 min for use at the point-of-care in the CW setting. </jats:p

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study

BACKGROUND: Quantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA. RESULTS: The reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance. CONCLUSIONS: Bayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens