Development and evaluation of a Luminex xTAG assay for sulfonamide resistance genes in Escherichia coli and Salmonella isolates.

Clinically occurring sulfonamide resistance in gram-negative bacteria is codified by several sul genes, mostly associated with the mobilized genetic elements named integrons, and integrons are frequently found in plasmids. There are four sul genes (sul1, sul2, sul3 and sul4) that encode resistance to sulfonamides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of all four sul genes and related Class 1 integrons (int1) in Escherichia coli and Salmonella isolates. The limits of detection ranged from 10 to 1000 copies/µL of input purified plasmid DNA. Forty-one bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for sul genes than conventional PCR. It indicated that the xTAG-multiplex PCR is a convenient method for rapid identification of sul genes.

Rapid detection of three rabbit pathogens by use of the Luminex x-TAG assay.

BACKGROUND: Domestic rabbits especially New Zealand white rabbits play an important role in biological research. The disease surveillance and quality control are essential to guarantee the results of animal experiments performed on rabbits. Rabbit hemorrhagic disease virus, rabbit rotavirus and Sendai virus are the important pathogens that needed to be eliminated. Rapid and sensitive method focus on these three viruses should be established for routine monitoring. The Luminex x-TAG assay based on multiplex PCR and fluorescent microsphere is a fast developing technology applied in high throughput detection. Specific primers modified with oligonucleotide sequence/biotin were used to amplify target fragments. The conjugation between oligonucleotide sequence of the PCR products and the MagPlex-TAG microspheres was specific without any cross-reaction, and the hybridization products could be analyzed using the Luminex 200 analyzer instrument. Recombinant plasmids were constructed to estimate the detection limit of the viruses. Furthermore, 40 clinical samples were used to evaluate the efficiency of this multiplex PCR based Luminex x-TAG assay. RESULTS: According to the results, this new method showed high specificity and good stability. Assessed by the recombinant plasmids, the detection limit of three viruses was 100copies/µl. Among 40 clinical specimens, 18 specimens were found positive, which was completely concordant with the conventional PCR method. CONCLUSIONS: The new developed Luminex x-TAG assay is an accurate, high throughput method for rapid detection of three important viruses of rabbits.

Comparison of Luminex xTAG® RVP fast assay and real time RT-PCR for the detection of respiratory viruses in adults with community-acquired pneumonia.

Community-acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT-PCR (rtRT-PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT-PCR (P = 0.3), and 2.7% for IFA (2.7%) (P < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT-PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT-PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs.

Diagnostic performance of the Luminex xTAG gastrointestinal pathogens panel to detect rotavirus in Ghanaian children with and without diarrhoea.

BACKGROUND: Rotavirus is one of the leading causes of childhood diarrhoea worldwide. The highest disease burden is seen in resource-constrained settings of sub-Saharan Africa. Recently, commercial multiplex PCR panels proved their accuracy to diagnose infectious gastroenteritis in Europe and the USA. However, data on their performance using samples from tropical regions in general and to detect rotavirus in particular remains scant. We aimed to analyse the diagnostic performance of the Luminex xTAG gastrointestinal pathogens panel, a multiplex PCR, to detect rotavirus in stool samples from Ghanaian children. METHODS: A total of 682 stool samples were collected in the Ashanti region of Ghana between 2007 and 2008. Of these, 341 were from cases (children with diarrhoea), and another 341 from controls (children without diarrhoea). All samples were analysed using the Luminex xTAG assay and compared to a rotavirus quantitative reverse-transcription PCR (reference assay). Rotavirus reference assay positive samples were P and G genotyped by sequencing the rotavirus VP4 and VP7 genes. RESULTS: Overall agreement between the Luminex xTAG and the reference assay was excellent (kappa 0.93). The sensitivity and specificity was 88.2 % (95 % confidence interval [CI] 78.2-94.1) and 100 % (95 % CI 99.2-100), respectively. Of 76 rotavirus reference assay positive samples, 64 were successfully genotyped and the Luminex xTAG assay was able to detect all rotavirus genotypes present in the study. CONCLUSION: The Luminex xTAG assay proved a sensitive and highly specific tool to detect rotavirus and may aid clinicians and public health authorities in the diagnosis and surveillance of rotavirus.

Comparative Evaluation of Luminex xTAG® Gastrointestinal Pathogen Panel and Direct-From-Stool Real-Time PCR for Detection of C. difficile Toxin tcdB in Stool Samples from a Pediatric Population.

Detection of Clostridioides difficile toxins in patients with gastroenteritis has increasingly been accomplished through the use of enteric multiplex syndromic panels. Comparisons of the performance of these panels to both direct-from-stool (DFS) and culture-enriched stools followed by polymerase chain reaction (PCR) methods in pediatric populations are limited. Here, we compare the performance of the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) to our DFS in-house real-time PCR (DFS RT-PCR) assay for the detection of C. difficile toxin gene, tcdB, using 2641 stool specimens collected from children enrolled in the Alberta Provincial Pediatric EnTeric Infection Team (APPETITE) study in Alberta, Canada. We used culture enrichment followed by in-house RT-PCR to resolve discordant results between the two assays. We found excellent agreement (k = 0.89) between the GPP and our DFS RT-PCR assay: the positive percent agreement between the two assays was 97%, and the negative percent agreement was 99%. GPP, a multi-analyte platform can easily be implemented into a routine diagnostic laboratory for detecting enteric pathogens including C. difficile.

Resolving discordant CYP2D6 genotyping results in Thai subjects: platform limitations and novel haplotypes.

Aim: Several CYP2D6 Luminex xTAG genotype calls were identified as inconsistent or suspicious among Thai subjects and further characterized to identify the root causes. Material & methods: Forty-eight subjects were followed-up with long-range-PCR, quantitative copy number assays and/or Sanger sequencing. Results: Most of the Luminex-duplication calls were either negative or had hybrid structures involving CYP2D6*36 in various configurations. Ten samples were inaccurately called as CYP2D6*2, *29 or *35 alleles. Sequencing revealed three novel haplotypes, CYP2D6*142, *143 and *144 of which two are nonfunctional. Conclusion: The Luminex platform produced a relatively high number of false genotype calls for Thai subjects. Our findings underscore the need for the systematic characterization of the CYP2D6 locus in diverse populations and rigorous platform validation.

The Complex Co-infections of Multiple Porcine Diarrhea Viruses in Local Area Based on the Luminex xTAG Multiplex Detection Method.

The large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since 2010, resulting in great damage to the pig industry. However, multiple infections have contributed to the outbreak of the disease and also resulted in great difficulties in diagnosis and control of the disease. Thus, a Luminex xTAG multiplex detection method, which was more sensitive and specific than general multiplex PCR method, was developed for the detection of 11 viral diarrhea pathogens, including PKoV, PAstV, PEDV, PSaV, PSV, PTV, PDCoV, TGEV, BVDV, PoRV, and PToV. To investigate the prevalence of diarrhea-associated viruses responsible for the outbreaks, a total of 753 porcine stool specimens collected from 9 pig farms in Shanghai during 2015-2018 were tested and the pathogen spectrums and co-infections were analyzed. As a result, PKoV, PAstV and PEDV were most commonly detected viruses in diarrheal pigs with the rate of 38.65% (291/753), 20.32% (153/753), and 15.54% (117/753), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 28.42%. Infection pattern of the viral diarrhea pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Furthermore, the surveillance confirmed that variant enteropathogenic viruses were leading etiologic agents of porcine diarrhea, either mono-infection or co-infections of PKoV were common in pigs in Shanghai, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status, suggesting that controlling porcine diarrhea might be more complex than previously thought. The study provides a better understanding of diarrhea viruses in piglets, which will aid in better preventing and controlling epidemics of viral porcine diarrhea.

CYP2D6 Predicts Plasma Donepezil Concentrations in a Cohort of Thai Patients with Mild to Moderate Dementia.

PURPOSE: Donepezil, a drug frequently used to treat dementia, is mainly metabolized by cytochrome P450 2D6 (CYP2D6). This study investigated the relationships between CYP2D6 genotype and activity scores as well as predicted phenotype of plasma donepezil concentrations in 86 Thai dementia participants. MATERIALS AND METHODS: CYP2D6 was genotyped using bead-chip technology (Luminex xTAG® v.3). Steady-state trough plasma donepezil concentrations were measured using high-performance liquid chromatography. RESULTS: Sixteen genotypes were found but the most frequent genotypes detected among our participants were CYP2D6*10/*10 (27.9%) and *1/*10 (26.7%). One-third of the participants had an activity score of 1.25 which predicted that they were normal metabolizers. The overall median (interquartile range) of plasma donepezil concentration was 51.20 (32.59-87.24) ng/mL. Normal metabolizers (NMs) had lower plasma donepezil concentrations compared to intermediate metabolizers (IMs) (41.15 (28.44-67.65) ng/mL vs 61.95 (35.25-97.00) ng/mL). Multivariate analysis showed that CYP2D6 activity score (r2 = 0.50) and the predicted phenotype (independent of dose) could predict the plasma donepezil concentration (r2 = 0.49). CONCLUSION: Plasma donepezil concentration in NMs was lower compared to IMs. Additional studies with larger sample size and use of next-generation sequencing as well as its outcomes are warranted to confirm the benefit of using pharmacogenetic-guided treatment for donepezil.

Development of a Luminex xTAG Assay for the Rapid Detection of Five Aminoglycoside Resistance Genes Both in Staphylococci and Enterococci.

Resistance to aminoglycoside antibiotics is now common in pathogenic bacteria, making treatment of infections difficult. The rapid spread of resistance is mainly related to the dissemination of genes encoding aminoglycoside-modifying enzymes (AMEs). Staphylococci and enterococci are opportunistic human pathogens capable of causing a wide range of infections. Isolates from clinical cases are often found to be resistant to aminoglycosides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of five prevalent aminoglycoside resistance genes in staphylococci and enterococci, including aac(6')-Ie-aph(2″)-Ia, aph(3')-IIIa, ant(4')-Ia, ant(9)-Ia, and ant(6)-Ia. The limit of detection ranged from 10 to 1000 copies/µL of input purified plasmid DNA. Twenty-two bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for AME genes than conventional PCR. It indicated that the xTAG-multiplex PCR method is a high-throughput tool for rapid identification of AME genes.

CYP2D6 genotype analysis of a Thai population: platform comparison.

The highly polymorphic CYP2D6 gene locus leads to a wide range of enzyme activity. Since there are limited data for Thai, the major aim was to investigate CYP2D6 genetic variation in a large Thai population (n = 920). CYP2D6 genotyping was performed using four different platforms. Genotype call rates of the Luminex xTAG® and AmpliChip CYP450 test were 96.5% and 87.4%, respectively. Based on Luminex xTAG® data, the most common alleles and genotypes were *1 0 (49.6%), *1 (24.6%), *2 (10.8%), *5 (6.7%), *41 (6.5%) and *1/*10 (23.9%), *10/*10 (21.5%), *2/*10 (9.4%), *5/*10 (6.9%), *10/*41 (5.7%), respectively. Challenges and limitations of the platforms evaluated are discussed. These data add to our knowledge regarding interethnic variability in CYP2D6 activity and contribute to improving drug therapy in the Thai.

Simultaneous detection of 4 prototypic rat parvoviruses using the luminex xTAG assay in laboratory animal health monitoring.

There are currently four rat parvoviruses including Kilham rat virus (KRV), Toolans H-1 parvovirus (H-1virus), rat parvovirus type 1a (RPV-1a) and rat minute virus (RMV). Virus detection methods are commonly based on conventional PCR - agarose gel electrophoresis or serological assay methods These methods are both time-consuming and lack specificity. In this study, we developed a bead array xTAG assay for the simultaneous detection and discrimination of four rat parvoviruses. The detection limits ranged from 100 to 1000 copies/µL of input purified plasmid DNA. We examined 50 clinical specimens and 15 facal samples by xTAG assay and conventional PCR. The results showed a high consistency except for several weak positive infections. It demonstrated that the xTAG-multiplex PCR method is specific, sensitive and suitable for high throughput platforms for rat parvovirus screening of clinical samples and contaminated biological materials.

Development of a molecular assay for the detection of Cucumber mosaic virus and the discrimination of its subgroups I and II.

A nucleic acid based test for the detection of the economically important plant virus Cucumber mosaic virus (CMV) based on the Luminex xTAG technology was developed. This technology has the advantage of allowing the simultaneous detection of various targets. Applying this method, we prove the presence of CMV in general and differentiate between its two subgroups I and II for which significant differences concerning severity of symptoms and virulence have been reported. For the development of the test procedure the coat protein gene sequences of 29 CMV isolates were cloned, sequenced and classified into subgroups. Sequences from GenBank were used to design primers. Additionally, a subgroup specific ELISA was conducted for comparison. This work is part of a project which aims to develop a test for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material.

Comparison of the Luminex xTAG Respiratory Viral Panel Fast v2 Assay With Anyplex II RV16 Detection Kit and AdvanSure RV Real-Time RT-PCR Assay for the Detection of Respiratory Viruses.

BACKGROUND: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay. METHODS: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay. RESULTS: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1-100% of the specimens. The agreement levels were relatively low (94.1-97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases). CONCLUSIONS: The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus.

Comparison of the Luminex xTAG Respiratory Viral Panel Fast v2 Assay With Anyplex II RV16 Detection Kit and AdvanSure RV Real-Time RT-PCR Assay for the Detection of Respiratory Viruses

BACKGROUND: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay. METHODS: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay. RESULTS: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1–100% of the specimens. The agreement levels were relatively low (94.1–97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases). CONCLUSIONS: The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus.