Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Screening asthmatics for atopic status using the ALergy EXplorer (ALEX2) macroarray.

BACKGROUND: Screening asthma patients for atopy facilitates management. Since 2010, the core biomarker for screening asthma subjects for atopic status has been the qualitative Phadiatop. multi-aeroallergen screen. A more quantitative macroarray, the Allergy Explorer (ALEX2), shows promise as an alternative. OBJECTIVE: The study's goal was to examine the pros and cons of the use of ALEX2 in the screening of asthma patients for atopic status. METHODS: We evaluated the atopic (IgE-sensitization) status in asthmatic Amish and Hutterite farm children using the ImmunoCAP and ALEX2 assays in Phadiatop equivocal and positive subjects. RESULTS: All 42 asthmatic children were analyzed by Phadiatop and total serum IgE. Of these, 22 had a negative Phadiatop (<0.1 kUa/L) and total IgE <100 kU/L which defined them as non-atopic and they were excluded from ALEX2 testing. Of six children with equivocal Phadiatops (0.1-0.2 kUa/L-Group 1) and three children with a negative Phadiatop but total IgE >100 kUa/L (group 3), 44% (n = 4) had detectable IgE antibody by ALEX2 to mite, tree pollen, and other allergens not detected by Phadiatop, but confirmed by allergen-specific ImmunoCAP testing. In 11 Phadiatop positive subjects (>0.2 kUa/L-group 2), all but one were positive by ALEX2. IgE antibody specific for mold and rabbit aeroallergens matched their agricultural and pet exposure history. Three children were positive for IgE antibody to allergens in the profilin, nsLTP, or PR-10 cross-reactive protein families. CONCLUSION: Judicious use of ALEX2's enhanced specificity data not provided by the Phadiatop can aid in the interpretation of sensitization patterns and planning management of atopic asthmatics, but sensitization relevance must be confirmed by the patient's clinical history.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Identification of cry genes in Bacillus thuringiensis by multiplex real-time PCR.

Bacillus thuringiensis is an important bacterium of the group Bacillus cereus sensu lato due to its insecticidal properties. This microorganism has high genetic variability and its strains produce different Cry toxins, known as δ-endotoxins, which are mainly responsible for its toxic effect on insects that are agricultural pests or vector human diseases. Each strain can express a variety of cry genes, out of a total of 789 cry genes described so far. The detection of these genes is very important to characterize strains, as they may indicate their toxic potential. Several methods have been used to characterize B. thuringiensis strains, but one of the most common techniques is Polymerase Chain Reaction (PCR) from primers that detect the presence of cry genes. This technique has been optimized to make real-time multiplex quantitative PCR (qPCR) assays faster, more efficient, and safer, because the presence of three genes can be detected in a single reaction. In this work, a multiplex assay was developed to identify the presence of genes from the cry1A, cry1C, and cry1F families whose respective toxins are present in both bioinsecticides, and commercial transgenic plants used to control caterpillars. Specific primers were designed to identify the families of the cited genes and the system was validated with samples that were sequenced by next-generation sequencing (NGS). The system was implemented and used to characterize 214 strains. Of these, eight were submitted to conventional PCR, and the results matched, again validating the system. Thus, the application of the proposed technique allows the reliable evaluation through this system to detect the presence of the genes of the families cry1A, cry1C, and cry1F in samples of B. thuringiensis.

Feasibility of the ALEX multiplex platform in the diagnosis of nut allergy from a Mediterranean population.

BACKGROUND: ALEX multiplex platform has been recently commercialized but its clinical utility as quantitative technique respect to ImmunoCAP-singleplex as the reference method has not yet been confirmed on patients suffering from nut allergy and co-sensitization to different nuts. METHODS: 58 serum samples from patients with nut allergy from a Mediterranean population were assayed in parallel by ALEX-multiplex and ImmunoCAP-singleplex techniques. Patients were diagnosed based on clinical symptoms and positive skin prick tests (SPTs). The following whole extracts were compared between both techniques: walnut, hazelnut, peanut, almond, pistachio and sunflower seed; besides the recombinant Pru p 3. A qualitative and quantitative study was carried out. RESULTS: Both techniques had similar sensitivities respect to whole extracts from walnut, hazelnut and peanut as well as to Pru p 3 (p > 0.05). However for whole extracts from almond, pistachio and sunflower seed the sensitivity obtained by ALEX was much lower than ImmunoCAP (9.09 % vs 88.63 %; 14.81 vs 70.37 %; and 8.51 % vs 88.88 %; respectively). The concordance between both techniques showed only a substantial agreement for Pru p 3 (k = 0.791); moderate agreement for hazelnut and peanut (k = 0.550 and k = 0.544, respectively); fair agreement for walnut (k = 0.386) and poor agreement for almond, pistachio and sunflower seed (k < 0.2). Quantitative analysis showed that ImmunoCAP for walnut, peanut and sunflower seed had higher mean values than ALEX. Relationships were significant for all specific IgE levels except to for almond, pistachio and sunflower seed. CONCLUSIONS: ALEX platform is a suitable technique to patients with nut allergy from the Mediterranean area except to for those suffering from allergy to almond, pistachio and sunflower seed.

Development of a Singleplex Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Quantification.

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3'-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay's performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident.

Combining Allergen Components Improves the Accuracy of Peanut Allergy Diagnosis.

BACKGROUND: IgE to peanut often occurs in the absence of peanut allergy. Detection of allergen component specific IgE (sIgE) has improved diagnosis and birthed molecular allergen component arrays, in which sensitization to multiple allergen components can be measured simultaneously. OBJECTIVE: To improve the diagnostic utility of serology for peanut allergy, by mapping interactions of sIgE to multiple components and IgE functional characteristics. METHODS: A cohort of 100 children was studied, with a 60-children cohort employed for external validation. Levels of total IgE, sIgE to peanut, and peanut components were measured using singleplex ImmunoCAP and multiplex immuno solid-phase allergen chip (ISAC). Peanut IgE specific activity, avidity, and diversity were determined. Diagnostic modeling was performed using a Bayesian hierarchical model. RESULTS: Sensitization to the 112 allergens on ISAC (model 1) demonstrated the highest accuracy to diagnose peanut allergy (area under the curve [AUC] = 0.92). Sensitization to peanut components on ISAC (model 2) reported an AUC of 0.86 and on singleplex (model 3) an AUC of 0.92, which was greater than that of Ara h 2 sIgE alone (AUC = 0.90). Functional characteristics of peanut sIgE (model 4) reported an AUC of 0.89, which was greater than that of peanut sIgE (AUC = 0.75). Model 3 offered the highest predictive value and the second highest overall diagnostic accuracy. CONCLUSIONS: sIgE to a combination of allergen components (Ara h 1, 2, 3, and 6) is highly predictive of peanut allergy and superior to individual markers. Combining the functional characteristics of IgE was superior to peanut sIgE levels alone. These models can be applied in real time during clinical consultations using online calculators.

Development and performance evaluation of the first in-house multiplex rRT-PCR assay in Bangladesh for highly sensitive detection of SARS-CoV-2.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic is posing a great threat to global health and economy. Due to the lack of broad diagnostic setup, consistent reagent supply lines, and access to laboratory instruments and equipment, it is undoubtedly an enormous burden for developing countries to face the crisis. OBJECTIVES: To develop a cost-effective, reliable and sensitive multiplex assay for SARS-CoV-2 screening which would expand the testing capacities of a developing and low-income country like Bangladesh. STUDY DESIGN: Initially a singleplex and then a multiplex real-time reverse-transcriptase PCR assays were developed targeting 2 nucleocapsid genes of SARS-CoV-2, and the human RNase P gene as an internal control using laboratory-made mastermixes. Three sets of primer- probes were designed for each of the target genes and one set was optimized for the final reaction set-up. Limit of detection, cross-reactivity and reproducibility were checked in order to assess the sensitivity and specificity of the assays, and validation was done using clinical specimens. RESULTS: Clinical evaluation of the new assays using 240 nasopharyngeal swabs showed 100 % sensitivity, specificity, and accuracy in detecting SARS-CoV-2 infection in human. Equal efficiency and concordant results were observed between the singleplex and multiplex approaches. Notably, the kit was able to detect SARS-CoV-2 RNA at very low concentration upto 5 copies/reaction. CONCLUSION: This is the first locally developed multiplex rRT-PCR kit in Bangladesh providing rapid and low-cost screening of COVID-19 which would be valuable for infection prevention and clinical management in the perspective of Bangladesh.

Validation study of a new chemiluminescent singleplex IgE assay in a set of Italian allergic rhinitis patients.

BACKGROUND: The measurement of specific IgE to allergenic extracts and molecules in patients with allergic rhinitis (AR) is crucial for a precise diagnosis and further immunotherapy. Companies providing in vitro diagnostic methods in allergology continuously strive for the optimization and modernization of such methods. A new generation of automated allergy tests based on chemiluminescence detection and paramagnetic microparticles is now available, with possible advantages in sample volume, cost-effectiveness and avoidance of sample-related interference. OBJECTIVES: To test whether sIgE antibody levels obtained with a new singleplex chemiluminescent method have a good agreement with the corresponding results obtained with a "gold standard" test. METHODS: We tested sera from 368 AR patients. Specific IgE sera levels (kU/L) to a comprehensive panel of 15 allergen extracts and 6 molecules were tested with ImmunoCAP® (Thermo Fisher Scientific Inc, Phadia AB, Uppsala, Sweden) and NOVEOS™ (HYCOR® Biomedical, Garden Grove, CA, USA). We evaluated the qualitative and quantitative performance of the new NOVEOS system in matching the outcome of ImmunoCAP to each of the examined allergens. RESULTS: In relation to ImmunoCAP, the overall diagnostic sensitivity and specificity of sIgE tests with NOVEOS were 90.8% (95% CI = 88.6-92.7) and 96.2% (95% CI = 93.9-97.8), respectively. These values were higher when only molecules were considered (sensitivity = 98.7% [95% CI = 96.4%-99.7%]; specificity = 94.2% [95% CI = 88.4%-97.6%]) and lower when only extracts were considered (sensitivity = 87.6% [95% CI = 84.7%-90.2%]; specificity = 97% [95% CI = 94.4%-98.6%]). Spearman's correlation between the data set of both methods for a ≥ 0.1 kU/L cut-off was 0.84 (p < .001). CONCLUSIONS: The new singleplex NOVEOS system presented good results for qualitative and quantitative comparisons when testing specific serum IgE antibodies against a range of 21 allergens. This novel immunoassay system using only 4 µl of sample per test appears to be robust and reliable and can, therefore, be used as an aid in allergy diagnosis.

Advances in IgE Testing for Diagnosis of Allergic Disease.

Since its discovery in 1967, IgE antibody detection in skin and blood has identified a state of allergic sensitization and served as a necessary but not sufficient risk factor that requires objective symptoms to make the definitive diagnosis of human allergic disease. More recently, quantitative IgE antibody levels in serum against allergenic extracts, molecules, and epitopes have pushed its application into more accurately identifying the specificity of the allergic response for targeting immunotherapy, predicting allergic symptom severity after allergen exposure, and attempting to distinguish tolerance from food allergy. This review examines new in vivo and in vitro developments in the design, performance, interference, and application of the methods used to identify allergic sensitization. The increasing accepted applications of molecular allergen and allergen epitope-based IgE antibody measurements, especially as applied to food allergy diagnosis and management, are highlighted as state-of-the-art advances. Despite these major advances in allergic sensitization documentation, their ultimate value requires integration by the clinician with the patient's history and pretest probability of disease.

Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors.

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).

Comparison of the performance of Skin Prick and ISAC Tests in the diagnosis of allergy.

Summary: The recent European Union and Italian regulations in the matter of in vivo test could strongly impact on current diagnostic approach, increasing the usage of in vitro tests in daily clinical practice. We evaluated 506 patients with both skin prick test and a microarray system (ImmunoCAP ISAC 112). The overall evaluation between ImmunoCAP® ISAC vs SPT showed a moderate agreement (k=0.509, 95% C.I. 0.480-0.540, SE: 0.016) considering both aeroallergens and food allergens. When we considered the concordant results (double-positive plus double-negatives), the agreement ranged from 69% to 80% for pollen allergens, between 74% and 76% for dust mites, and between 74% and 93% for animal epithelia. In the case of food allergens, the accordance was pretty lower, accounting values ranging from 67% to 86%. ISAC testing identified from 22% to 26% more cases than SPTs in peach and nuts hyper-sensitivity. In 2.8% of the control group, the ISAC-test failed to detect an allergy sensitization caused by dust mite, shrimp, Anisakis, or seed storage proteins. Multiplex testing is more than a promising tool for more precise and comprehensive profiling of allergic patients and can be considered as a second-line approach, after the anamnesis, in the diagnosis of allergic diseases.

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens.

BACKGROUND: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. RESULTS: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml). CONCLUSIONS: Based on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104-5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Differentiation of Francisella tularensis Subspecies and Subtypes.

The highly infectious and zoonotic pathogen Francisella tularensis is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe Francisella tularensis subsp. novicida, there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes. The methods currently available to genotype F. tularensis cannot conclusively identify the associated subpopulation without using time-consuming testing or complex scoring matrices. To address this need, we developed both single and multiplex quantitative real-time PCR (qPCR) assays that can accurately detect and identify the hypervirulent F. tularensis subsp. tularensis subtype A.I, the virulent F. tularensis subsp. tularensis subtype A.II, F. tularensis subsp. holarctica (also referred to as type B), and F. tularensis subsp. mediasiatica, as well as opportunistic F. tularensis subsp. novicida from each other and near neighbors, such as Francisella philomiragia, Francisella persica, and Francisella-like endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific F. tularensis subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified F. tularensis targets provides clade confirmation and informative strain-specific details. Application of these qPCR- and sequencing-based detection assays will provide an improved capability for molecular typing and clinical diagnostics, as well as facilitate the accurate identification and differentiation of F. tularensis subpopulations during epidemiological investigations of tularemia source outbreaks.

Evaluation of a new multiplex assay for allergy diagnosis.

BACKGROUND: Molecular allergy has significantly improved the quality of allergy diagnosis; however, the positioning of singleplex and multiplex assays in the diagnostic algorithm is still a matter of debate. METHODS: Aim of the study was to test the analytical performance of the recently commercialized Allergy Explorer-ALEX® in a selected population (105 allergic patients and 15 negative controls), comparing it with the reference ImmunoCAP® method and with skin prick test (SPT). RESULTS: Inter-assay qualitative comparison showed a substantial agreement between ALEX® and SPT (k = 0.64). A substantial agreement between ALEX® and ImmunoCAP® was shown on the detection of IgE to extracts (k = 0.64 for inhalants and k = 0.51 for food allergens), whereas a higher agreement was shown on detection of molecular components (k = 0.92 for inhalants and k = 0.72 for food allergens). Quantitative comparison showed a poor correlation between ALEX® and ImmunoCAP®. CONCLUSION: The simultaneous detection of both extracts and molecular components with ALEX® assay can potentially overcome some of the major limitations of the multiplex assay currently in use. However, before using ALEX® as routine method, the analytical performance (in particular for extracts) needs to be further investigated on a larger scale.

Precision medicine allergy immunoassay methods for assessing immunoglobulin E sensitization to aeroallergen molecules.

Molecular-based allergy diagnosis for the in vitro assessment of a patient immunoglobulin E (IgE) sensitization profile at the molecular level uses allergen molecules (also referred to as allergen components), which may be well-defined, highly purified, natural allergen components or recombinant allergens. Modern immunoassay methods used for the detection of specific IgE against aeroallergen components are either singleplex (such as the fluorescence enzyme immunoassay with capsulated cellulose polymer solid-phase coupled allergens, the enzyme-enhanced chemiluminescence immunoassay and the reversed enzyme allergosorbent test, with liquid-phase allergens), multiparameter (such as the line blot immunoassay for defined partial allergen diagnostics with allergen components coating membrane strips) or multiplex (such as the microarray-based immunoassay on immuno solid-phase allergen chip, and the two new multiplex nanotechnology-based immunoassays: the patient-friendly allergen nano-bead array, and the macroarray nanotechnology-based immunoassay used as a molecular allergy explorer). The precision medicine diagnostic work-up may be organized as an integrated "U-shape" approach, with a "top-down" approach (from symptoms to molecules) and a "bottom-up" approach (from molecules to clinical implications), as needed in selected patients. The comprehensive and accurate IgE sensitization molecular profiling, with identification of the relevant allergens, is indicated within the framework of a detailed patient's clinical history to distinguish genuine IgE sensitization from sensitization due to cross-reactivity (especially in polysensitized patients), to assess unclear symptoms and unsatisfactory response to treatment, to reveal unexpected sensitizations, and to improve assessment of severity and risk aspects in some patients. Practical approaches, such as anamnesis molecular thinking, laboratory molecular thinking and postmolecular anamnesis, are sometimes applied. The component-resolved diagnosis of the specific IgE repertoire has a key impact on optimal decisions making for prophylactic and specific immunotherapeutic strategies tailored for the individual patient.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin.

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.

Predicting the Functional Types of Singleplex and Multiplex Eukaryotic Membrane Proteins via Different Models of Chou's Pseudo Amino Acid Compositions.

Given a membrane protein sequence, how can we identify its type, particularly when a query protein may have the multiplex character, i.e., simultaneously exist at two or more different types. However, most of the existing predictors or methods can only be used to deal with the single-type or "singleplex" membrane proteins. Actually, multiple-type or "multiplex" membrane proteins should not be ignored because they usually posses some unique biological functions worthy of our special notice. In this study, three different models were developed, which have the ability to deal with the systems containing both singleplex and multiplex membrane proteins. The overall success rate thus obtained was 0.6440, indicating that the study may become a very useful high-throughput tool in identifying the functional types of membrane proteins.