Standard Multiplex Polymerase Chain Reaction Research Articles

Antimicrobial prescribing after rapid influenza PCR implementation in the emergency department

IntroInfluenza shares common symptoms with bacterial pneumonia, which may result in unnecessary antibiotic prescriptions in the emergency department (ED) when the diagnosis is unknown. Rapid influenza polymerase chain reaction (PCR) tests have reduced turnaround times compared to standard multiplex PCR respiratory panels allowing for earlier diagnosis, which may improve antimicrobial stewardship outcomes in the ED. This study aims to compare antibiotic and antiviral use before and after deployment of the rapid influenza PCR in the ED. MethodsThis single-center, retrospective, cohort study included pediatric and adult patients discharged from the ED with a positive influenza test using a standard multiplex PCR respiratory panel (January 2017 – July 2019) or rapid PCR (July 2019 – February 2020). The primary endpoint was number of antibiotic prescriptions pre- and post-implementation of the rapid influenza PCR in the ED. Secondary endpoints included number of antiviral prescriptions, duration of antimicrobial therapy, test turnaround time, ED length of stay, 30-day readmission, and adverse events. A multivariable logistic regression evaluated patient factors associated with antimicrobial prescribing. ResultsA total of 620 positive influenza results were identified with 280 patients (standard multiplex PCR = 33; rapid PCR = 247) meeting inclusion criteria. Patients were less likely to be prescribed antibiotics (39.4% vs 8.9%, OR 0.15, 95% CI 0.067–0.34) and more likely to be prescribed antivirals (24.2% vs 61.1%, OR 4.92, 95% CI 2.13–11.34) with the rapid influenza PCR. Rapid influenza PCR significantly reduced ED length of stay (4.9 vs 3.4 h, p < 0.01) and test turnaround time (27 h vs 3.5 h, p < 0.01). Patients at high risk for complications associated with influenza were more likely to be prescribed antiviral therapy (22.7% vs 67.8%, OR 7.16, 95% CI 2.52–20.40). Based on the regression analysis conducted, asthma, (OR 3.5, 95% CI 1.48–8.26), immunosuppression (OR 9.6, 95% CI 1.18–78.2), and age <5 years old (OR 3.1, 95% CI 1.80–5.45) were predictors of antiviral prescribing. ConclusionImplementation of a rapid influenza PCR in the ED reduced antibiotic use and optimized antiviral therapy for patients with influenza including those at higher risk of complications.

Circulating Respiratory Syncytial Virus Genotypes and Genetic Variability of the G Gene during 2017 and 2018/2019 Seasonal Epidemics Isolated from Children with Lower Respiratory Tract Infections in Daejeon, Korea

BackgroundRespiratory syncytial virus (RSV) is a major pathogen causing respiratory tract infections in infants and young children. The aim of this study was to confirm the genetic evolution of RSV causing respiratory infections in children at Daejeon in Korea, through G gene analysis of RSV-A and RSV-B strains that were prevalent from 2017 to 2019.MethodsPediatric patients admitted for lower respiratory tract infections at The Catholic University of Korea Daejeon St. Mary's Hospital in the 2017 and 2018/2019 RSV seasonal epidemics, who had RSV detected via multiplex polymerase chain reaction (PCR) were included. The nucleic acid containing RSV-RNA isolated from each of the patients' nasal discharge during standard multiplex PCR testing was stored. The G gene was sequenced and phylogenetic analysis was performed using MEGA X program and the genotype was confirmed.ResultsA total of 155 specimens including 49 specimens from 2017 and 106 specimens from 2018-2019 were tested. The genotype was confirmed in 18 specimens (RSV-A:RSV-B = 4:14) from 2017 and 8 specimens (RSV-A:RSV-B = 7:1) from 2018/2019. In the phylogenetic analysis, all RSV-A type showed ON1 genotype and RSV-B showed BA9 genotype.ConclusionRSV-B belonging to BA9 in 2017, and RSV-A belonging to ON1 genotype in 2018/2019 was the most prevalent circulating genotypes during the two RSV seasons in Daejeon, Korea.

PCR GeneScan and Heteroduplex Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) or consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium. The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan and heteroduplex analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Rapid method for Growth Hormone Receptor exon 3 delete (GHRd3) SNP genotyping from archival human placental samples

Analysis of archival samples from cohorts of pregnant women may be key to discovering prognosticators of stillbirth and pregnancy/perinatal complications. Growth hormone (GH) and its receptor (GHR) are pivotal in feto-placental development and pregnancy maintenance. We report a rapid, optimized method for genotyping the GHR full-length versus exon 3-deleted isoform (GHRd3). TaqMan single nucleotide polymorphism (SNP) genotyping proved superior to standard multiplex polymerase chain reaction (PCR) in allele detection and GHR genotyping from archived samples, including those with poor genomic deoxyribonucleic acid quality/quantity such as formalin fixed, paraffin embedded, blood, and serum. Furthermore, this assay is suitable for high through put 96 or 384-well plate quantitative PCR machines with automated genotype calling software. The TaqMan genotyping assay can increase the data obtained from precious archival human samples.

Rapid method for growth hormone receptor exon 3 delete (GHRd3) SNP genotyping from archival human placental samples.

Analysis of archival samples from cohorts of pregnant women may be key to discovering prognosticators of stillbirth and pregnancy/perinatal complications. Growth hormone (GH) and its receptor (GHR) are pivotal in feto-placental development and pregnancy maintenance. We report a rapid, optimized method for genotyping the GHR full-length versus exon 3-deleted isoform (GHRd3). TaqMan single nucleotide polymorphism (SNP)genotyping proved superior to standard multiplex polymerase chain reaction (PCR) in allele detection and GHR genotyping from archived samples, including those with poor genomic deoxyribonucleic acid quality/quantity such as formalin fixed, paraffin embedded, blood, and serum. Furthermore, this assay is suitable for high through put 96 or 384-well plate quantitative PCR machines with automated genotype calling software. The TaqMan genotyping assay can increase the data obtained from precious archival human samples.

Screening for AZFc partial deletions in Dravidian men with nonobstructive azoospermia and oligozoospermia.

Dravidians are the predominant population residing in South India with a diverse genetic structure. Considering various genetic discoveries taking place today, it is evident that deletions in the AZFc region are the most common cause of severe spermatogenic failure (SSF) in various populations studied. However, it is significant to note that there is a paucity of scientific literature on AZFc subdeletion screening among the Dravidian population. To investigate the prevalence and association of AZFc subdeletion patterns among Dravidian men with nonobstructive azoospermia (NOA) and oligozoospermia. A population of 354 subjects, including 120 patients with NOA, 109 with oligozoospermia, and 125 normal male controls, were screened using locus-specific sequence tag site markers. We found 21 (9.17%) patients with classical AZF deletion, while no deletions were observed in controls. After excluding the samples with AZF deletions, the remaining 208 infertile and 125 control samples were screened for partial AZFc deletions using a standardized multiplex polymerase chain reaction and on analysis revealed that 13 (6.25%) of the infertile samples possessed gr/gr subdeletions and 15 (7.21%) of the infertile samples possessed b2/b3 subdeletions. Six (4.8%) of the normal samples were found to carry gr/gr subdeletions and two (1.6%) had b2/b3 deletions. The b1/b3 deletion was not observed in any of the patient and control samples screened. Our finding shows that there is a strong association between b2/b3 subdeletion and SSF in the Dravidian population (odds ratio, 4.78; 95% confidence interval 1.07-21.26) (p=0.018). Further studies, including gene copy typing for DAZ and CDY genes and a comprehensive haplogrouping analysis, are recommended in a large and well-selected patient group to elude the genetic mechanism behind this association.

Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

Immunoglobulin/T-cell receptor clonality diagnostics

Clonality testing in lymphoid malignancies has become technically relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for Ig/T-cell receptor (TCR) gene analysis. Expertise with clonality diagnostics and knowledge about the biology of Ig/TCR recombination are essential for correct interpretation of the Ig/TCR clonality data. Several immunobiologic and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this review. Furthermore, the need to integrate the molecular data with that from (hemato-)pathology, and preferably also flowcytometric immunophenotyping for appropriate interpretation, is discussed. Such an interactive, multidisciplinary diagnostic model guarantees integration of all available data to reach the most reliable diagnosis.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Multi-Node Graphs: A Framework for Multiplexed Biological Assays

Multiplex polymerase chain reaction (PCR) is an extension of the standard PCR protocol in which primers for multiple DNA loci are pooled together within a single reaction tube, enabling simultaneous sequence amplification, thus reducing costs and saving time. Potential cost saving and throughput improvements directly depend on the level of multiplexing achieved. Designing reliable and highly multiplexed assays is challenging because primers that are pooled together in a single reaction tube may cross-hybridize, though this can be addressed either by modifying the choice of primers for one or more amplicons, or by altering the way in which DNA loci are partitioned into separate reaction tubes. In this paper, we introduce a new graph formalism called a multi-node graph, and describe its application to the analysis of multiplex PCR scalability. We show, using random multi-node graphs that the scalability of multiplex PCR is constrained by a phase transition, suggesting fundamental limits on efforts to improve the cost-effectiveness and throughput of standard multiplex PCR assays. In particular, we show that when the multiplexing level of the reaction tubes is roughly theta(log (sn)) (where s is the number of primer pair candidates per locus and n is the number of loci to be amplified), then with very high probability we can 'cover' all loci with a valid assignment to one of the tubes in the assay. However, when the multiplexing level of the tube exceeds these bounds, there is no possible cover and moreover the size of the cover drops dramatically. Simulations using a simple greedy algorithm on real DNA data also confirm the presence of this phase transition. Our theoretical results suggest, however, that the resulting phase transition is a fundamental characteristic of the problem, implying intrinsic limits on the development of future assay design algorithms.

Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis

We developed a method for the detection of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers. The method is based on the quantitative analysis of the products of standard multiplex polymerase chain reaction (PCR) from 18 different exons of the dystrophin gene, and is designated "QM-PCR." We detected deletions of one or more exons by standard multiplex PCR in DMD/BMD patients in 14 of 18 families examined (77.7%). The same deletions were readily demonstrated by QM-PCR in nine of 14 mothers (64.3%) and in another six of 22 possible carriers in these families. In five families where deletions were detectable in DMD/BMD patients, the mothers did not exhibit any deletions in their peripheral blood (35.7%). We obtained evidence for germinal mosaicism in at least two of these families and confirmed carrier identification by haplotype analysis using CA repeat polymorphisms at the 5' and 3' ends of the dystrophin gene. Furthermore, analysis of 17 coded DNA samples from normal females and obligatory carriers by QM-PCR showed that this technique could directly identify carriers of deletions in any of 18 different exons of the dystrophin gene. Its application in combination with existing techniques is expected to significantly improve the accuracy of carrier diagnosis in many families, and it may also be applicable to families in which pedigree and polymorphism information is insufficient for carrier diagnosis.