Singleplex Assays Research Articles

Comparison of an automated microfluidic immunoassay technology (BioIC, lab-on-chips) and ImmunoCAP assay. Lab-on-chips as a tool for specific IgE (sIgE) detection.

IntroductionThe measurement of clinically important specific IgE (sIgE) antibody is pivotal for both diagnosis and management of allergy. Two methods may be distinguished depending on the number of antigens tested simultaneously: singleplex and multiplex. BioIC is a multiplex, advanced, automated microfluidic immunoassay system enabling simultaneous sIgE measurement against multiple allergens. ImmunoCAP is a singleplex assay for sIgE detection and gold standard method for diagnosis of allergy.AimTo compare and validate the diagnostic capability of a multiplex sIgE assay – BioIC assay with a singleplex ImmunoCAP assay.Material and methodsUsing both BioIC assay and ImmunoCAP assay, the sIgE level in serum samples from 20 allergic disease patients with respect to 33 allergens (16 inhalant allergens, 16 food allergens and 1 contact allergen) was measured. Receiver operating characteristic (ROC) area under the curve (AUC), qualitative and semi-quantitative comparisons were performed to compare both sIgE measurement methods using statistical analyses.ResultsROC AUC analysis showed similar sensitivity and specificity of BioIC assay and ImmunoCAP assay. In qualitative analysis, the negative and positive agreements were 100% equal for each allergen. Spearman’s rank correlation coefficients identified very high positive correlations between two assays for all tested allergens (p < 0.001).ConclusionsThe BioIC showed agreement with ImmunoCAP assay. Sensitivity and specificity of both assays are similar, thus they showed similar diagnostic performance. However, careful interpretation of obtained results is necessary in clinical applications because of methodological differences between these two systems.

Parasite-derived circulating microRNAs as biomarkers for the detection of human Schistosoma japonicum infection.

Novel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

Vitamin D and Inflammatory Markers in Children with Sickle Cell Disease

Background: Vitamin D, in addition to its role in calcium and bone homeostasis, is a multifunctional regulator of inflammation and of innate and adaptive immune responses. Deficiencies are prevalent in sickle cell disease. We conducted a randomized double- blind active-controlled clinical trial comparing high- (100,000 IU/month) and low-dose (12,000 IU/month) oral vitamin D3 in 62 children with sickle cell disease to reduce respiratory complications including infection, asthma exacerbation and acute chest syndrome (ViDAS trial: Vitamin D for sickle cell respiratory complications; NCT01443728). A secondary aim was to examine the effects of vitamin D on immune function and inflammation. Methods: Using stored serum samples collected from subjects at steady state, we examined hsCRP (single-plex assay) and key cytokines and markers of vascular activation (20-plex immunoassay) at baseline and year-1 in a subset of 36 subjects with HbSS (high-dose: n=17; low-dose: n=19), mean age 7.8 years, 53% females and 67% on hydroxyurea. Immune markers included IL1β, IL1RA, IL2, IL4, IL10, IL18, IL22, IFNγ, TNFα, TNFβ, sFasL, CXCL1, CXCL8 (IL8), PDGFBB, VEGFA, serpin E1 (PAI1), sICAM1, VCAM1, TGFα and TGFβ. Raw immune data were transformed using Box-Cox transformations and standardized; markers with persistently skewed (non-Gaussian) distributions after transformation were dichotomized for analysis. Continuous immune variables were analyzed by repeated measures ANOVA (unadjusted) and ANCOVA (adjusted for baseline covariates of age, sex and baseline total 25-hydroxyvitamin D3 and D2). Dichotomized biomarkers were analyzed using generalized linear mixed-effects models. Secondary analyses stratified on hydroxyurea status were also pursued. For these exploratory biomarker discovery-focused analyses, all significance tests were performed 2-tailed without adjustment for multiple comparisons, with α=0.1 for main effects and 0.2 for interactions. Results: Independent of vitamin D3 dose, serum concentrations of immune markers declined from baseline to the 1-year timepoint for the pro-inflammatory markers IL2, CXCL8 (IL8), IFNγ, TGFα, CXCL1 and PDGFBB and the counter-regulatory molecule, IL10. Among all subjects, interactions of dose group*time were found for IL2, sICAM1 and hsCRP; subjects on hydroxyurea additionally demonstrated dose group*time interactions for serpin E1 (PAI1), IFNγ and TNFα. Subjects on hydroxyurea in the high-dose group also demonstrated decreases over 1-year follow-up in serum IL2, serpin E1, IFNγ, TNFα, sICAM1 and hsCRP whereas serum levels of these immune markers increased among subjects in the low-dose group taking hydroxyurea. This crossover pattern in direction of the dose group*time interaction effect was also observed for the larger study population for sICAM1 and hsCRP. Table 1 shows the markers that tested significantly for main effects and/or interactions. Effects for the rest of the markers were not statistically significant. Conclusions: Our data support the immunomodulatory properties of vitamin D, with variable responses to high and low doses. Effects involved several salient immune and vascular markers for sickle cell disease, including IL2, serpin E1, IFNγ, TNFα, sICAM1 and hsCRP, and appeared to be more pronounced among subjects on high-dose vitamin D3 who were also taking hydroxyurea. Table 1 Disclosures Brittenham: Nestec, Inc.: Consultancy; Novartis International: Consultancy; Tesseract Health, Inc.: Consultancy; Vifor Fresenius Medical Care: Consultancy; Ambys Medicines: Consultancy; Dispersol Technologies: Consultancy; Rockwell Medical, Inc.: Consultancy.

128P - Multiplex chromogenic Immunohistochemistry (IHC) for spatial analysis of checkpoint-positive tumour infiltrating lymphocytes (TILs)

BackgroundSpatial localization of TILs has been shown to correlate with immunotherapy response. Chromogenic IHC (cIHC) enables assessment of lineage (eg, CD8) or immune checkpoint expression (eg, T-cell immunoglobulin and mucin-domain–containing-3 [TIM-3]). Codetection of lineage and checkpoint molecules may help to better inform responders to checkpoint inhibitors, but colocalization is challenging by cIHC. Fluorescence (fIHC) allows colocalization but not spatial assessment and is not an approved CDx platform. Consequently, CDxs have been limited to single-plex cIHC assays. We developed a multiplex cIHC platform that enables image analysis colocalization and spatial assessment of TILs. MethodsImage analysis of cIHC via HALO software was performed to detect colocalized CD8 and TIM-3. We compared % TIM-3+/CD8+ by cIHC to TIL multiparameter flow cytometry (MFC) on 10 procured renal cell carcinoma (RCC) samples. Pathologist annotation and a trained random forest machine-learning tissue classifier were used to determine spatial localization (tumor vs peritumoral vs nontumor) of CD8+ and TIM-3+/CD8+ TIL subsets. We also performed a validated fIHC assay, including CD8 and TIM-3, on 5 additional procured RCC samples. ResultsHigh concordance (r=0.92) was observed between % TIM3+/CD8+ TILs measured by cIHC and MFC. Image analysis showed that within the CD8+ population, median % TIM-3+/CD8+ TILs were primarily intratumoral (18.0%), with fewer in peritumoral (9.4%) and nontumoral (6.4%) regions (intratumoral vs peritumoral, and intratumoral vs nontumor, both P=0.015). TIM-3/CD8 coexpression detection appeared similar between cIHC and fIHC. ConclusionsWe developed a multiplex cIHC method that enables simultaneous quantitation of TIL subpopulations, checkpoint expression, and spatial analysis. cIHC, fIHC and MFC performed similarly in coexpression detection, but only multiplex cIHC enabled spatial localization. Specifications of this assay show promise for development as a CDx. Additional data will demonstrate the capability to substitute TIM-3 with other markers, suggesting an assay template for other lineage/target combinations. Editorial acknowledgementAmrita Dervan, MSc, and Jay Rathi, MA, of Spark Medica Inc, funded by Bristol-Myers Squibb. Legal entity responsible for the studyBristol-Myers Squibb. FundingBristol-Myers Squibb. DisclosureS. Ely: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. G. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. L. Menard: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. J. Yan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Fischer: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. B. Kakrecha: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. D. Locke: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. P. Patah: Shareholder / Stockholder / Stock options, Full / Part-time employment: Bristol-Myers Squibb. K. Urbanska: Full / Part-time employment: Bristol-Myers Squibb.

Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA

Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation – lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.

Development of conventional and real time PCR assays for rapid species authentication of mammalian cell lines commonly used in veterinary diagnostic laboratories

Mammalian cell lines are valuable tools in biomedical fields, with applications ranging from disease diagnosis to the production of biological reagents and vaccines. Here we report the development of new conventional (cPCR) and real time PCR (qPCR) assays for species identification of several mammalian kidney cell lines originated from swine, green monkey, hamster and bovine tissues that are extensively used in veterinary diagnostic laboratories. The PCR primers and probes were selected from highly conserved mitochondrial genes and analyzed in silico by nucleotide BLAST in the National Center for Biotechnology Information (NCBI) website to ensure target specificity. The assays were highly species-specific and had no cross-reactivity against other tested cell lines originated from different mammalian species. Assay sensitivity (limit of detection; LOD) was determined using serial dilutions of cell line DNA as template. The estimated LODs were between 2.95 and 48 pg (picogram) DNA/assay for cPCR, and between 1.5 × 10−3 and 4.8 × 10−2 pg DNA/assay for qPCR. Multiplex qPCR assays were developed for simultaneous detection of up to three species in a single assay. The multiplex qPCR assays exhibited the same sensitivity as the corresponding singleplex assays with the exception of the green monkey species that demonstrated a 10–100 fold decline in the sensitivity. Contamination of swine cells was detected in one of the rabbit cell lines. The contamination was further confirmed by Sanger and Next-Generation sequencing.

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 μL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-μm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays—mostly high signal to noise ratio of extended chemiluminescent imaging—are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR

Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krškopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.

Interleukin-6 is associated with mortality and neuropsychiatric outcomes in antiretroviral-naïve adults in Rakai, Uganda.

Serum interleukin-6 (IL-6) and D-dimer have been associated with multiple adverse outcomes in HIV-infected (HIV+) individuals, but their association with neuropsychiatric outcomes, including HIV-associated neurocognitive disorder (HAND) and depression, headaches, and peripheral neuropathy have not been investigated. Three hundred ninety-nine HIV+ antiretroviral therapy (ART)-naïve adults in Rakai, Uganda, were enrolled in a longitudinal cohort study and completed a neurological evaluation, neurocognitive assessment, and venous blood draw. Half of the participants had advanced immunosuppression (CD4 count < 200cells/μL), and half had moderate immunosuppression (CD4 count 350-500cells/μL). All-cause mortality was determined by verbal autopsy within 2years. HAND was determined using Frascati criteria, and depression was defined by the Center for Epidemiologic Studies-Depression (CES-D) scale. Neuropathy was defined as the presence of > 1 neuropathy symptom and > 1 neuropathy sign. Headaches were identified by self-report. Serum D-dimer levels were determined using ELISA and IL-6 levels using singleplex assays. Participants were 53% male, mean age 35 + 8years, and mean education 5 + 3years. Participants with advanced immunosuppression had significantly higher levels of IL-6 (p < 0.001) and a trend toward higher D-dimer levels (p = 0.06). IL-6 was higher among participants with HAND (p = 0.01), with depression (p = 0.03) and among those who died within 2years (p = 0.001) but not those with neuropathy or headaches. D-dimer did not vary significantly by any outcome. Systemic inflammation as measured by serum IL-6 is associated with an increased risk of advanced immunosuppression, all-cause mortality, HAND, and depression but not neuropathy or headaches among ART-naïve HIV+ adults in rural Uganda.

Urinary TERT promoter mutations as non-invasive biomarkers for the comprehensive detection of urothelial cancer

BackgroundRecurrent mutations in the promoter of the telomerase reverse transcriptase (TERT) gene (C228T and C250T) detected in tumours and cells shed into urine of urothelial cancer (UC) patients are putative biomarkers for UC detection and monitoring. However, the possibility of detecting these mutations in cell-free circulating DNA (cfDNA) in blood and urine, or DNA from urinary exfoliated cells (cellDNA) with a single-gene sensitive assay has never been tested in a case-control setting. MethodsWe developed a single-plex assay (UroMuTERT) for the detection of low-abundance TERT promoter mutations. We tested 93 primary and recurrent UC cases and 94 controls recruited in France (blood, urine samples and tumours for the cases), and 50 primary UC cases and 50 controls recruited in Portugal (urinary exfoliated cell samples). We compared our assay with urine cytology. FindingsIn the French series, C228T or C250T were detected in urinary cfDNA or cellDNA in 81 cases (87·1%; 95% CI 78·6–93·2), and five controls (Specificity 94·7%; 95%CI 88·0–98·3), with 98·6% (95% CI 92·5–99·96) concordance in matched tumours. Detection rate in plasma cfDNA among cases was 7·1%. The UroMuTERT sensitivity was (i) highest for urinary cfDNA and cellDNA combined, (ii) consistent across primary and recurrent cases, tumour stages and grades, (iii) higher for low-risk non-muscle invasive UC (86·1%) than urine cytology (23·0%) (P < 0·0001) and (iv) 93·9% when combined with cytology. In the Portuguese series – the sensitivity and specificity for detection of UC with urinary cellDNA was 68·0% (95% CI 53·3–80·5) and 98·0% (95% CI 89·3–100·0). InterpretationTERT promoter mutations detected by the UroMuTERT assay in urinary DNA (cfDNA or cellDNA) show excellent sensitivity and specificity for the detection of UC, significantly outperforming that of urine cytology notably for detection of low-grade early stages UC. FundFrench Cancer League; French Foster Research in Molecular Biology and European Commission FP7 Marie Curie COFUND.

A Multiplex Quantitative Reverse Transcription Polymerase Chain Reaction Assay for the Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pilocytic Astrocytomas.

Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR). We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs. The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time. The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.

Development and evaluation of a one-step multiplex real-time TaqMan\xae RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples

BackgroundEquine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens.MethodsA one-step multiplex TaqMan® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined.ResultsThe multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, > 90% for G3 VP7 and > 99% for G14 VP7, respectively) and high overall agreement (> 98%) compared to conventional RT-PCR and sequencing.ConclusionsThis new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field.

Development and validation of a multiplexed drug level assay in support of combination biologics therapy clinical studies

Clinical development of biotherapeutics for combination therapy requires monitoring the concentrations of both drugs in biological samples. Traditionally, two assays are required to measure drug levels one at a time, which poses challenges in sample management, data reporting, and cost. The Meso Scale Discovery (MSD®) U-PLEX™ platform provides a simple and flexible way to create custom multiplex ligand binding assays (LBAs). We developed and fully validated a two-plex assay on the U-PLEX platform where two therapeutic monoclonal antibodies (mAbs) in Merck’s pipeline, which we call MK-A and MK-B in this manuscript, can be measured simultaneously in one sample. Our results demonstrated that the multiplexed pharmacokinetic (PK) assay has performances, including accuracy, precision, and cross-reactivity, that meet requirements in regulatory guidance. Furthermore, results of MK-A from the multiplex assay are comparable to results from a previously validated MK-A single-plex assay with 80% of samples tested in both assays having concentration differences < 30% relative to the mean of the two measurements. The multiplex assay was used to support a phase I MK-A/MK-B combination therapy clinical study and generated results consistent with historical MK-A monotherapy PK data. The ability to measure both biotherapeutics in a multiplexed assay is beneficial in that it improves consistency and efficiency while reduces sample volume and cost. With the number of combination therapies increasing in development, multiplexed assays can potentially have wide applications in biologics bioanalysis.

Etiology and characteristics of community-acquired pneumonia in an influenza epidemic period

PurposeThe etiology of community-acquired pneumonia (CAP) in hospital patients is often ambiguous due to the limited pathogen detection. Lack of a microbiological diagnosis impairs precision treatment in CAP. MethodsSpecimens collected from the lower respiratory tract of 195 CAP patients, viruses were measured by the Single-plex real-time PCR assay and the conventional culture method was exploited for bacteria. ResultsAmong the 195 patients, there were 46 (23.59%) pure bacterial infections, 20 (10.26%) yeast infections, 32 (16.41%) pure viral infections, 8 (4.10%) viral-yeast co-infections, and 17 (8.72%) viral-bacterial co-infections. The two most abundant bacteria were Acinetobacter baumannii and klebsiella pneumoniae, whereas the most common virus was influenza A. ConclusionsNon-influenza respiratory microorganisms frequently co-circulated during the epidemic peaks of influenza, which easily being ignored in CAP therapy. In patients with bacterial and viral co-infections, identifying the etiologic agent is crucial for patient’s therapy.

Staphylococcus aureus and methicillin resistance detection directly from pediatric samples using PCR assays with differential cycle threshold values for corroboration of methicillin resistance

Staphylococcus aureus is a major human pathogen, causing a variety of nosocomial and community-acquired infections. While S. aureus usually grows well, there are situations where it cannot be isolated in culture, such as patients who have received prior antimicrobial therapy. There are commercially available tests for molecular identification of S. aureus and methicillin resistance; however, they often have limited utility due to restrictive specimen requirements, lack of data in pediatric populations and issues with specificity for methicillin resistance detections. Our objective was to evaluate the performance of laboratory-developed PCR assays that detect S. aureus and methicillin resistance directly from various specimen types. We developed two real-time PCR assays: 1) a singleplex assay targeting the nucA gene and 2) a multiplex PCR assay (mecA/SCC-orf PCRs) that detects the mecA gene and the conjunction region where SCCmec elements insert into the genome. A total of 538 pediatric specimens, including specimens from the lower respiratory tract (n = 149), abscess/wounds (n = 245), tissue and body fluids (n = 144), were tested and the results compared with culture and susceptibility testing. The nucA PCR is sensitive and specific for detection of S. aureus when compared with culture with an overall agreement of 93.1% and sensitivity and specificity of 93.5% and 93.0%, respectively. Among those culture-confirmed and nucA PCR positive specimens (n = 145), concordance between mecA/SCC-orf PCRs, using cycle threshold values for corroboration, and conventional methods was 98.6% and the sensitivity and specificity were 97.3% and 100%, respectively. The assays' performance suggests they are rapid, reliable tools to detect and differentiate between methicillin susceptible and methicillin resistant S. aureus in our pediatric patient population providing diagnostic impact when used in conjunction with culture.

Evaluation of a new multiplex assay for allergy diagnosis

BackgroundMolecular 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. MethodsAim 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). ResultsInter-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®. ConclusionThe 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.

Simultaneous detection of coconut lethal yellowing phytoplasmas (group 16SrIV) by real-time PCR assays using 16Sr- and GroEL-based TaqMan probes

Lethal yellowing (LY) represents a serious threat to coconut in Mexico, Central America and the Caribbean islands. It is caused by a phytoplasma of group 16SrIV, and particularly subgroups -A and -D have been the major phytoplasmas affecting coconut palms grown in the southeast of Mexico. Therefore, is important to reliably detect the 16SrIV group of phytoplasmas not only for diagnosis purposes but also to improve the understanding of pathogen-plant-vector pathosystems. The 16S ribosomal operon genes have been particularly used as the targets for probes and primers for universal phytoplasma detection. However, its conservative nature has imposed the search of non-ribosomal genes to support a finer characterization and differentiation of phytoplasmas, particularly those closely related and one alternative has been the GroEL gene for the specific detection of subgroups 16SrIV-A and 16SrIV-D. Sensitive, rapid and reliable singleplex and duplex assays were developed based on real-time PCR (TaqMan) targeting both the GroEL gene and 16S rRNA. The assay performed with higher sensitivity compared to conventional nested-PCR when analysing DNA extracts obtained from LY symptom bearing palms. The LY 16S TaqMan probe assays amplified DNA from both subgroups, while the LY GroEL TaqMan probe assay only amplified DNA from subgroup 16SrIV-A. The duplex TaqMan probe assay is a novel option for the simultaneous detection and quantification of LY phytoplasmas of 16Sr subgroups -A and -D, which are the phytoplasmas that predominantly affect palms in Mexico.

The development of a multiplex serological assay for avian influenza based on Luminex technology.

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.

Comparison of FTD® respiratory pathogens 33 and a singleplex CDC assay for the detection of respiratory viruses: A study from Cameroon

Introduction: This study compares the detection of 14 common respiratory viruses by two different real-time reverse transcription polymerase chain reaction (rRT-PCR) methods: in house singleplex tests developed by the Centers for Disease Control and Prevention and the commercially available Fast Track Diagnostic (FTD®) Respiratory Pathogens 33 multiplex test. Methods: A total of 217 nasopharyngeal swabs were analyzed using CDC singleplex rRT-PCR and FTD® Respiratory Pathogens 33 multiplex assays, for the detection of 14 respiratory viruses. Results: The results showed that 179/217 (82.5%) samples were positive with the singleplex method and 183/217 (84.3%) with the FTD® Respiratory Pathogens 33 multiplex test. Excellent or satisfactory agreement was obtained for all viruses (k > 0.6) except Parainfluenzavirus 4 (k = 0.24) and influenza B (k = 0.51). Conclusion: Although the multiplex FTD kits were more expensive than the singleplex assay, the FTD kits yielded rapid results in a shorter timeframe, increasing efficiency of diagnosis.

A Duo 4-Plex Real Time PCR for Detection of Eight Tick-Borne Zoonoses in Kenya

Ticks harbor multiple pathogens, most of which can be transmitted to humans. The ensuing zoonoses display non-specific symptoms that make definitive diagnosis difficult. We report here the development and evaluation of multiplex real time polymerase chain reaction (qPCR) assays for eight tick-borne zoonoses (TBZ). The assays were organized in duo formats of 4-plex each. Format 1 was optimized for Anaplasma phagocytophilum, Coxiella burnetii, Borrelia burgdoferi and Ehrlichia chaffeensis. Format 2 was optimized for Rickettsia species (spp.), Bartonella spp., Borrelia spp. other than B. burgdoferi and Babesia spp. Synthetic plasmids were used to show that the assays can specifically detect all target sequences in the same reaction tube. Assays were assayed eight times to determine assay performance and the limit of detection was determined as the lowest plasmid concentration that was amplified for all the targets. Standard curves of threshold cycle (Ct) versus copy numbers were generated and used to determine linearity and efficiency of the assays. Pairwise comparison of singleplex and multiplex assays was done using Bland-Altman plots. Prevalence was calculated as overall percentage of positive patients to each TBZ tested Assay 1 had a limit of detection of 2 copy numbers for all targets. Assay 2 was less sensitive and on average had a limit of detection of 18 gene copies. In replicate tests, both assays had intra-assay variation of less than two cycles. Multiplex assays performance was comparable to respective singleplex assays. Evaluation of 512 clinical samples collected between 2008 and 2016 from acute febrile illness patients attending hospitals in different counties in Kenya revealed a 20% prevalence of tick-borne pathogens comprising B. burgdorferi (6%), non B. burgdorferi Borrelia spp. (3%), C. burnetii (5%), A. phagocytophilum (5%), Rickettsia spp. (2%), E. chaffeensis (0.8%), Bartonella spp. (0.8%), and Babesia spp. (0.4%). The high analytical sensitivity suggests potential for the duo 4-plex qPCR for detection of common TBZ.