Clinical comparison of three SARS-CoV-2 nucleic acid amplification tests for routine diagnostic testing.

Background: Cycle threshold (Ct) values from SARS-CoV-2 nucleic acid amplification tests have been used to estimate viral load for treatment decisions. Additionally, there is a need for high-throughput testing, consolidating a variety of assays on one random-access analyzer. Objectives: In this study, the clinical performance of the Alinity m SARS-CoV-2, RealTime SARS-CoV-2, and GeneXpert Xpress SARS-CoV-2/Flu/RSV assays was assessed. Methods: Alinity precision and detection rates were evaluated using a dilution series of the Alinity m SARS-CoV-2 positive control. In a retrospective study, 7 remnant external quality assessment (EQA) specimens and 200 remnant nasopharyngeal swab specimens (100 positive and 100 negative) were tested in the three assays. Results: Alinity had 100 % detection rate at 50 copies/mL and high reproducibility (Ct value coefficient of variation ≤3.1 %). All three assays correctly detected positive and negative EQA samples with comparable Ct values (max difference 2.38) and high linearity. In patient samples, positive percent agreement was 95 % (95 % CI 89-98 %) and negative percent agreement was 100 % (95 % CI 96-100 %) for Alinity, compared to the other two assays. Four specimens detected on Alinity m but not RealTime or Xpert had Ct values above 40. Assay results were highly correlated (r ≥ 0.94). Ct values (after addition of 10 unread cycles to the reported Ct of RealTime) were comparable across the three assays. Conclusions: Alinity m had high precision and accuracy and Ct values comparable to those of the RealTime and Xpert assays. The assays could be used interchangeably, with no need for adjustment of patient management decisions based on Ct values from each assay.

Benchmarking medical laboratory performance: survey validation and results for Europe, Middle East, and Africa.

OBJECTIVES: Medical laboratory performance is a relative concept, as are quality and safety in medicine. Therefore, repetitive benchmarking appears to be essential for sustainable improvement in health care. The general idea in this approach is to establish a reference level, upon which improvement may be strived for and quantified. While the laboratory community traditionally is highly aware of the need for laboratory performance and public scrutiny is more intense than ever due to the SARS-CoV-2 pandemic, few initiatives span the globe. The aim of this study was to establish a good practice approach towards benchmarking on a high abstraction level for three key dimensions of medical laboratory performance, generate a tentative snapshot of the current state of the art in the region of Europe, Middle East, and Africa (EMEA), and thus set the stage for global follow-up studies. METHODS: The questionnaire used and previously published in this initiative consisted of 50 items, roughly half relating to laboratory operations in general with the other half addressing more specific topics. An international sample of laboratories from EMEA was approached to elicit high fidelity responses with the help of trained professionals. Individual item results were analyzed using standard descriptive statistics. Dimensional reduction of specific items was performed using exploratory factor analysis and assessed with confirmatory factor analysis, resulting in individual laboratory scores for the three subscales of "Operational performance", "Integrated clinical care performance", and "Financial sustainability". RESULTS: Altogether, 773 laboratories participated in the survey, of which 484 were government hospital laboratories, 129 private hospital laboratories, 146 commercial laboratories, and 14 were other types of laboratories (e.g. research laboratories). Respondents indicated the need for digitalization (e.g. use of IT for order management, auto-validation), automation (e.g. pre-analytics, automated sample transportation), and establishment of formal quality management systems (e.g. ISO 15189, ISO 9001) as well as sustainably embedding them in the fabric of laboratory operations. Considerable room for growth also exists for services provided to physicians, such as "Diagnostic pathways guidance", "Proactive consultation on complex cases", and "Real time decision support" which were provided by less than two thirds of laboratories. Concordantly, the most important kind of turn-around time (TAT) for clinicians, sample-to-result TAT, was monitored by only 40% of respondents. CONCLUSIONS: Altogether, the need for stronger integration of laboratories into the clinical care process became apparent and should be a main trajectory of future laboratory management. Factor analysis confirmed the theoretical constructs of the questionnaire design phase, resulting in a reasonably valid tool for further benchmarking activities on the three aimed-for key dimensions.

Benchmarking diagnostic laboratory performance: Survey results for Germany, Austria, and Switzerland.

BACKGROUND AND AIMS: The need for patient safety through consistent diagnostic performance has increasingly been brought into focus during the last two decades. Around the globe operational efficiency of diagnostic laboratories plays a key role in satisfying this need, which has impressively been shown during the recent months of the SARS-CoV­2 pandemic. On a global level, however, there has been a lack to collate and benchmark data for diagnostic laboratories. The goals of this study were to design and pilot a questionnaire addressing key aspects of diagnostic laboratory management. METHODS: The questionnaire was designed using an iterative process and taking into consideration information that could be extracted from the literature, author experience and feedback from informal focus groups of laboratory professionals. The resulting tool consisted of 50 items, either relating to general information or more specifically addressing the topics of "operational performance", "integrated clinical care performance", and "financial sustainability". A limited number of laboratories were surveyed to be able to further improve the newly developed tool and motivate the global laboratory community to participate in further benchmarking activity. RESULTS AND CONCLUSION: Altogether, 65 laboratories participated in the survey, 42 were hospital laboratories and 23 were commercial laboratories. Potential for further improvement and standardization became apparent across the board, e.g. use of IT for order management, auto-validation, or turn-around time (TAT) monitoring. Notably, a gap was identified regarding services provided to physicians, in particular "reflexive test suggestions", "proactive consultation on complex cases", and "diagnostic pathways guidance", which were only provided by about two thirds of laboratories. Concordantly, within-laboratory TAT (Lab TAT) was monitored by about 80% of respondents, while sample-to-result TAT, which is arguably the TAT most relevant to clinicians, was only monitored by 32% of respondents. Altogether, the need for stronger integration of the laboratory into the clinical care process became apparent and should be a main trajectory of future laboratory management.

IgE Epitopes of the House Dust Mite Allergen Der p 7 Are Mainly Discontinuous and Conformational.

Background: Several studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7. Methods: Recombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1-P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes. Results: rDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7. Conclusion and Clinical Relevance: IgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

BACKGROUND: In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects. METHODS: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively. RESULTS: House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10. CONCLUSION: IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed.

Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.