Assessing Different PCR Master Mixes for Ultrarapid DNA Amplification: Important Analytical Parameters.

The basic principles of ultrafast plasmonic PCR have been promulgated in the scientific and technological literature for over a decade. Yet, its everyday diagnostic utility remains unvalidated in pre-clinical and clinical settings. Although the impressive speed of plasmonic PCR reaction is well-documented, implementing this process into a device form compatible with routine diagnostic tasks has been challenging. Here, we show that combining careful system engineering and process control with innovative and specific PCR biochemistry makes it possible to routinely achieve a sensitive and robust "10 min" PCR assay in a compact and lightweight system. The critical analytical parameters of PCR reactions are discussed in the current instrument setting.

Pharmacogenetic Expression of CYP2C19 in a Pediatric Population.

Genetic variability in CYP2C19 may be associated with both lack of efficacy and toxicity of drugs due to its different metabolic status based on the presence of particular alleles. This literature review summarizes current knowledge relative to the association or treatment adaptation based on CYP2C19 genetics in a pediatric population receiving drugs metabolized by CYP2C19, such as voriconazole, antidepressants, clopidogrel and proton pump inhibitors. Additionally, we also presented one of the approaches that we developed for detection of variant alleles in the CYP2C19 gene. A total of 25 articles on PubMed were retained for the study. All studies included pediatric patients (age up to 21 years) having benefited from an assessment of CYP2C19. CYP2C19 poor and intermediate metabolizers exhibit a higher trough plasma concentration of voriconazole, and PPIs compared to the rapid and ultra-rapid metabolizers. The pharmacogenetic data relative to CYP2C19 and clopidogrel in the pediatric population are not yet available. CYP2C19 poor metabolizers have a higher trough plasma concentration of antidepressants compared to the rapid and the ultra-rapid metabolizers. Modification of allele-specific PCR through the introduction of artificial mismatch is presented. CYP2C19 genotyping remains a powerful tool needed to optimize the treatment of children receiving voriconazole, PPIs, and anti-depressants.

Importance of Adequate qPCR Controls in Infection Control.

Respiratory screening assays lacking Sample Adequacy Controls (SAC) may result in inadequate sample quality and thus false negative results. The non-adequate samples might represent a significant proportion of the total performed tests, thus resulting in sub-optimal infection control measures with implications that may be critical during pandemic times. The quantitative sample adequacy threshold can be established empirically, measuring the change in the frequency of positive results, as a function of the numerical value of "sample adequacy". Establishing a quantitative threshold for SAC requires a big number/volume of tests to be analyzed in order to have a statistically valid result. Herein, we are offering for the first time clear clinical evidence that a subset of results, which did not pass minimal sample adequacy criteria, have a significantly lower frequency of positivity compared with the "adequate" samples. Flagging these results and/or re-sampling them is a mitigation strategy, which can dramatically improve infection control measures.

Sample Adequacy Control (SAC) Lowers False Negatives and Increases the Quality of Screening: Introduction of "Non-Competitive" SAC for qPCR Assays.

Sample Adequacy Control (SAC) has critical analytical, clinical and epidemiological value that increases confidence in a negative test result. The SAC is an integral qPCR assay control, which ensures that all pre-analytical and analytical steps are adequate for accurate testing and reporting. As such, a negative SAC with a negative result on pathogen screen specifies that the result should be reported as inconclusive instead of negative. Despite this, many regulatory approved tests do not incorporate SAC into their assay design. Herein, we emphasize the universal value of SAC and offer for the first time, a simple technical strategy to introduce non-competitive SAC which does not interfere with the limit of detection for the screened pathogen. Integration of SAC can provide key benefits towards identifying, isolating, quarantining and contact tracing infected individuals and in turn can improve worldwide efforts in infection control.

Maximizing confidence in a negative result: Quantitative sample adequacy control.

Quantitative PCR (qPCR) is a leading screening tool, permitting rapid detection of pathogens and the maintenance of effective infection control programs. Unfortunately, qPCR assays frequently do not incorporate Sample Adequacy Control (SAC). A SAC controls for the quantity, quality and adequacy of the specimen. Without SAC, the confidence in a negative result remains questionable and the efficacy of screening is compromised. Ultimately, the exclusion of SAC from qPCR may result in false negative results. One should consider SAC to be an integral critical type of laboratory control; addressing diverse analytical problems, such as sample adequacy, sample processing and assay inhibition. Following distribution of cycle threshold values (Cq) of Influenza A positive results and Cq values of SAC, obtained from nasopharyngeal swabs, we showed that the confidence in a negative result cannot be guaranteed in the presence of a weak positive SAC signal (late Cq values). Herein, we explain why widespread inclusion of sample adequacy control in routine screening is blocked. A protocol and methods for SAC threshold establishment are offered.

Laboratory-developed test for detection of acute Clostridium difficile infections with the capacity for quantitative sample normalization.

We describe a laboratory-developed test intended for the detection of acute Clostridium difficile infections (CDI) with the capacity for quantitative sample normalization. The test is based on the detection of the tcdB gene. However, this biomarker is also present among people without symptoms, implying that individuals with diarrhea, not caused by C. difficile may nonetheless test positive. Therefore, clinical diagnosis based on this format of testing can be challenging. In order to improve diagnostic assays capability, tcdB-based quantification methods were suggested as a potential solution, however they did not increase clinical specificity. We report methodology for a dual biomarker monitoring (total bacterial load and tcdB assay), allowing for the calculation of the relative presence of tcdB in the total bacterial population in the tested samples. We believe that this approach is clinically relevant to current assays and can improve CDI testing algorithms.

Detection and Isolation of Clostridium difficile Asymptomatic Carriers During Clostridium difficile Infection Outbreaks: An Exploratory Study.

During 4 Clostridium difficile infection outbreaks, unit-wide screening of 114 patients led to detection and isolation of 15 (13%) C. difficile asymptomatic carriers. Carriage prevalence varied between outbreaks, from 0% to 29% (P = .004). Isolating carriers was not associated with significantly shorter outbreak durations, compared with historical controls.

Rectal swab screening assays of public health importance in molecular diagnostics: Sample adequacy control.

Rectal swabs are routinely used by public health authorities to screen for multi-drug resistant enteric bacteria including vancomycin-resistant enterococci (VRE) and carbapenem-resistant enterobacteriaceae (CRE). Screening sensitivity can be influenced by the quality of the swabbing, whether performed by the patient (self-swabbing) or a healthcare practitioner. One common exclusion criterion for rectal swabs is absence of "visible soiling" from fecal matter. In our institution, this criterion excludes almost 10% of rectal swabs received in the microbiology laboratory. Furthermore, over 30% of patients in whom rectal swabs are cancelled will not be re-screened within the next 48h, resulting in delays in removing infection prevention measures. We describe two quantitative polymerase chain reaction (qPCR)-based assays, human RNAse P and eubacterial 16S rDNA, which might serve as suitable controls for sampling adequacy. However, lower amounts of amplifiable human DNA make the 16s rDNA assay a better candidate for sample adequacy control.

Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines.

Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

Assay for estimating total bacterial load: relative qPCR normalisation of bacterial load with associated clinical implications.

Relative microorganism abundance is a parameter describing biodiversity, referring to how common a bacterial species is within the total bacterial flora. Anal, rectal, skin, mucal, and respiratory swabs are typical clinical samples where knowledge of relative bacterial abundance might make distinction between asymptomatic carriers and symptomatic cases. Assays trying to measure total bacterial load are usually based on the amplification of universal segments of 16S rRNA genes. Previous assays were not adoptable to "direct" PCR protocols, and/or they were not compatible with hydrolysis-based detection. Using the latest summary of universal 16S sequence motifs present in literature and testing our design with 500 liquid and 50 formed stool samples, we illustrate the performance characteristics of a new 16S quantitative PCR (qPCR) assay, which addresses well-known technical problems, including a) positive priming reaction in the absence of intended target due to self-priming and/or mispriming of unintended targets; b) amplification bias due to nonoptimal primer/probe coverage; and c) too large amplicons for clinical qPCR. Stool swabs ranked into bins of different bacterial loads show significant correlation with threshold cycle values of our new assay. To the best of our knowledge, this is the first description of qPCR assay measuring individual differences of total bacterial load present in human stool.

Bim polymorphisms: influence on function and response to treatment in children with acute lymphoblastic leukemia.

PURPOSE: Corticosteroids induce apoptosis in the malignant lymphoid cells and are critical component of combination therapy for acute lymphoblastic leukemia (ALL). Several genome-wide microarray studies showed major implication of proapoptotic Bim in mediating corticosteroid-related resistance in leukemia cells. EXPERIMENTAL DESIGN: We investigated Bim gene polymorphisms and their association with childhood ALL outcome, and the mechanism underlying the observed finding. RESULTS: Lower overall survival (OS) was associated with Bim C29201T located in Bcl-2 homology 3 (BH3) domain (P = 0.01). An association remained significant in multivariate model (P = 0.007), was more apparent in high-risk patients (P = 0.004) and patients treated with dexamethasone (P = 0.009), and was subsequently confirmed in the replication patient cohort (P = 0.03). RNA analysis revealed that C29201T affects generation of γ isoforms (γ1) that lack proapoptotic BH3 domain. The phenotypic effect was minor suggesting the influence of additional factors that may act in conjunction with Bim genotype. Combined analysis with Mcl gene polymorphism (G-486T) revealed profound reduction in OS in individuals with both risk genotypes (P < 0.0005 in discovery and P = 0.002 in replication cohort) and particularly in high-risk patients (P ≤ 0.008). CONCLUSIONS: Increased expression of prosurvival Mcl1 and presence of Bim isoforms lacking proapoptotic function might explain marked reduction of OS in a disease and dose-dependent manner in ALL patients carrying Bim- and Mcl1-risk genotypes.

Significantly improved performance of a multitarget assay over a commercial SCCmec-based assay for methicillin-resistant Staphylococcus aureus screening: applicability for clinical laboratories.

Detection of the SCCmec gene is a common strategy for methicillin-resistant Staphylococcus aureus (MRSA) screening assays. However, if SCCmec is used alone, it may not be specific for detecting MRSA. Herein, we describe an in-house MSRA PCR-based screening assay involving detection of three targets (nuc, coa, and mecA). The assay is suitable for a wide range of real-time PCR platforms used in clinical microbiological laboratories. Performance characteristics of the in-house assay were significantly improved compared with the commercial assay, leading to an increase of positive predictive value by 40%. Compared with the bacterial culture, the sensitivity, specificity, and positive and negative predictive values of the in-house PCR assay were 100% (95% CI >83.2%), 99.2% (95% CI = 98.2% to 99.8%), 80% (95% CI = 59.3% to 93.2%), and 100% (95% CI >99.2%), respectively.

Host and pathogen factors for Clostridium difficile infection and colonization.

BACKGROUND: Clostridium difficile infection is the leading cause of health care-associated diarrhea, and the bacterium can also be carried asymptomatically. The objective of this study was to identify host and bacterial factors associated with health care-associated acquisition of C. difficile infection and colonization. METHODS: We conducted a 15-month prospective study in six Canadian hospitals in Quebec and Ontario. Demographic information, known risk factors, potential confounding factors, and weekly stool samples or rectal swabs were collected. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates to determine the genotype. Levels of serum antibodies against C. difficile toxins A and B were measured. RESULTS: A total of 4143 patients were included in the study; 117 (2.8%) and 123 (3.0%) had health care-associated C. difficile infection and colonization, respectively. Older age and use of antibiotics and proton-pump inhibitors were significantly associated with health care-associated C. difficile infection. Hospitalization in the previous 2 months; use of chemotherapy, proton-pump inhibitors, and H(2) blockers; and antibodies against toxin B were associated with health care-associated C. difficile colonization. Among patients with health care-associated C. difficile infection and those with colonization, 62.7% and 36.1%, respectively, had the North American PFGE type 1 (NAP1) strain. CONCLUSIONS: In this study, health care-associated C. difficile infection and colonization were differentially associated with defined host and pathogen variables. The NAP1 strain was predominant among patients with C. difficile infection, whereas asymptomatic patients were more likely to be colonized with other strains. (Funded by the Consortium de Recherche sur le Clostridium difficile.).

Fourteen-genome comparison identifies DNA markers for severe-disease-associated strains of Clostridium difficile.

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼ 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

DNA variants in region for noncoding interfering transcript of dihydrofolate reductase gene and outcome in childhood acute lymphoblastic leukemia.

PURPOSE: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A(-317) and C(-1610) alleles. Haplotype *1 was also associated higher DHFR expression. EXPERIMENTAL DESIGN: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript. RESULTS: Six polymorphisms were identified, of which five were single nucleotide polymorphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into five subtypes (*1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G(308)A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels. CONCLUSIONS: The study provides a new insight into DHFR regulatory variations predisposing to an event in ALL patients.

A protocol for the in vitro selection of specific oligonucleotide probes for high-resolution DNA typing.

The confident discrimination of nucleic acids that share a high degree of sequence identity is the major obstacle for the widespread applicability of multiplex DNA-based techniques. This diagnostic uncertainty originates in the insufficient specificity of hybridization, allowing cross-hybridization between unwanted probe-target combinations. Starting from a random mixture of oligonucleotides, we describe a protocol to selectively amplify the probes that bind to the target but not to the similar, unintended targets. The procedure involves five forward hybridizations to generate pools of probes with significant affinity, but not necessarily specificity, for the target. Specificity is then achieved during subtractive hybridization steps, where only probes having differential diagnostic performance are retained. Iterative hybridizations, cloning, sequencing and testing of the performance of selected probes can all be fully automated. Eight weeks are required for the full completion of a project composed of 40 probe-target pairs, even when targets share as much as 87% of sequence identity. While alternative, computer-assisted, rational oligonucleotide design may produce an uncertain outcome, the present protocol generates robust and specific probes suitable for a variety of multiplex, nucleic acid-based detection/typing platforms.