Discriminating Bacterial and Viral Infection Using a Rapid Host Gene Expression Test.

OBJECTIVES: Host gene expression signatures discriminate bacterial and viral infection but have not been translated to a clinical test platform. This study enrolled an independent cohort of patients to describe and validate a first-in-class host response bacterial/viral test. DESIGN: Subjects were recruited from 2006 to 2016. Enrollment blood samples were collected in an RNA preservative and banked for later testing. The reference standard was an expert panel clinical adjudication, which was blinded to gene expression and procalcitonin results. SETTING: Four U.S. emergency departments. PATIENTS: Six-hundred twenty-three subjects with acute respiratory illness or suspected sepsis. INTERVENTIONS: Forty-five-transcript signature measured on the BioFire FilmArray System (BioFire Diagnostics, Salt Lake City, UT) in ~45 minutes. MEASUREMENTS AND MAIN RESULTS: Host response bacterial/viral test performance characteristics were evaluated in 623 participants (mean age 46 yr; 45% male) with bacterial infection, viral infection, coinfection, or noninfectious illness. Performance of the host response bacterial/viral test was compared with procalcitonin. The test provided independent probabilities of bacterial and viral infection in ~45 minutes. In the 213-subject training cohort, the host response bacterial/viral test had an area under the curve for bacterial infection of 0.90 (95% CI, 0.84-0.94) and 0.92 (95% CI, 0.87-0.95) for viral infection. Independent validation in 209 subjects revealed similar performance with an area under the curve of 0.85 (95% CI, 0.78-0.90) for bacterial infection and 0.91 (95% CI, 0.85-0.94) for viral infection. The test had 80.1% (95% CI, 73.7-85.4%) average weighted accuracy for bacterial infection and 86.8% (95% CI, 81.8-90.8%) for viral infection in this validation cohort. This was significantly better than 68.7% (95% CI, 62.4-75.4%) observed for procalcitonin (p < 0.001). An additional cohort of 201 subjects with indeterminate phenotypes (coinfection or microbiology-negative infections) revealed similar performance. CONCLUSIONS: The host response bacterial/viral measured using the BioFire System rapidly and accurately discriminated bacterial and viral infection better than procalcitonin, which can help support more appropriate antibiotic use.

Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population.

OBJECTIVES: To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. STUDY DESIGN AND METHODS: Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. RESULTS: In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. CONCLUSIONS: Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Immune Profiling Panel: A Proof-of-Concept Study of a New Multiplex Molecular Tool to Assess the Immune Status of Critically Ill Patients.

BACKGROUND: Critical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting impaired immune response. In the field of diagnosis, a gap still remains in identifying the immune profile of critically ill patients in the intensive care unit (ICU). METHODS: A new multiplex immune profiling panel (IPP) prototype was assessed for its ability to semiquantify messenger RNA immune-related markers directly from blood, using the FilmArray System, in less than an hour. Samples from 30 healthy volunteers were used for the technical assessment of the IPP tool. Then the tool was clinically assessed using samples from 10 healthy volunteers and 20 septic shock patients stratified using human leukocyte antigen-DR expression on monocytes (mHLA-DR). RESULTS: The IPP prototype consists of 16 biomarkers that target the immune response. The majority of the assays had a linear expression with different RNA inputs and a coefficient of determination (R2) > 0.8. Results from the IPP pouch were comparable to standard quantitative polymerase chain reaction and the assays were within the limits of agreement in Bland-Altman analysis. Quantification cycle values of the target genes were normalized against reference genes and confirmed to account for the different cell count and technical variability. The clinical assessment of the IPP markers demonstrated various gene modulations that could distinctly differentiate 3 profiles: healthy volunteers, intermediate mHLA-DR septic shock patients, and low mHLA-DR septic shock patients. CONCLUSIONS: The use of IPP showed great potential for the development of a fully automated, rapid, and easy-to-use immune profiling tool. The IPP tool may be used in the future to stratify critically ill patients in the ICU according to their immune status. Such stratification will enable personalized management of patients and guide treatments to avoid secondary infections and lower mortality.

The influence of nucleotide sequence and temperature on the activity of thermostable DNA polymerases.

Extension rates of a thermostable, deletion-mutant polymerase were measured from 50°C to 90°C using a fluorescence activity assay adapted for real-time PCR instruments. Substrates with a common hairpin (6-base loop and a 14-bp stem) were synthesized with different 10-base homopolymer tails. Rates for A, C, G, T, and 7-deaza-G incorporation at 75°C were 81, 150, 214, 46, and 120 seconds(-1). Rates for U were half as fast as T and did not increase with increasing concentration. Hairpin substrates with 25-base tails from 0% to 100% GC content had maximal extension rates near 60% GC and were predicted from the template sequence and mononucleotide incorporation rates to within 30% for most sequences. Addition of dimethyl sulfoxide at 7.5% increased rates to within 1% to 17% of prediction for templates with 40% to 90% GC. When secondary structure was designed into the template region, extension rates decreased. Oligonucleotide probes reduced extension rates by 65% (5'-3' exo-) and 70% (5'-3' exo+). When using a separate primer and a linear template to form a polymerase substrate, rates were dependent on both the primer melting temperature (Tm) and the annealing/extension temperature. Maximum rates were observed from Tm to Tm - 5°C with little extension by Tm + 5°C. Defining the influence of sequence and temperature on polymerase extension will enable more rapid and efficient PCR.

Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments.

BACKGROUND: Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. METHODS: A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. RESULTS: The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. CONCLUSIONS: Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

Stopped-flow DNA polymerase assay by continuous monitoring of dNTP incorporation by fluorescence.

DNA polymerase activity was measured by a stopped-flow assay that monitors polymerase extension using an intercalating dye. Double-stranded DNA formation during extension of a hairpin substrate was monitored at 75°C for 2 min. Rates were determined in nucleotides per second per molecule of polymerase (nt/s) and were linear with time and polymerase concentration from 1 to 50 nM. The concentrations of 15 available polymerases were quantified and their extension rates determined in 50 mM Tris, pH 8.3, 0.5 mg/ml BSA, 2 mM MgCl2, and 200 µM each dNTP as well as their commercially recommended buffers. Native Taq polymerases had similar extension rates of 10-45 nt/s. Three alternative polymerases showed faster speeds, including KOD (76 nt/s), Klentaq I (101 nt/s), and KAPA2G (155 nt/s). Fusion polymerases including Herculase II and Phusion were relatively slow (3-13 nt/s). The pH optimum for Klentaq extension was between 8.5 and 8.7 with no effect of Tris concentration. Activity was directly correlated to the MgCl2 concentration and inversely correlated to the KCl concentration. This continuous assay is relevant to PCR and provides accurate measurement of polymerase activity using a defined template without the need of radiolabeled substrates.

Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.

BACKGROUND: High-resolution melting of PCR products is an efficient and analytically sensitive method to scan for sequence variation, but detected variants must still be identified. Snapback primer genotyping uses a 5' primer tail complementary to its own extension product to genotype the resulting hairpin via melting. If the 2 methods were combined to analyze the same PCR product, the residual sequencing burden could be reduced or even eliminated. METHODS: The 27 exons and neighboring splice sites of the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene were amplified by the PCR in 39 fragments. Primers included snapback tails for genotyping 7 common variants and the 23 CFTR mutations recommended for screening by the American College of Medical Genetics. After symmetric PCR, the amplicons were analyzed by high-resolution melting to scan for variants. Then, a 5-fold excess of H2O was added to each reaction to produce intramolecular hairpins for snapback genotyping by melting. Each melting step required <10 min. Of the 133 DNA samples analyzed, 51 were from CFTR patient samples or cell lines. RESULTS: As expected, the analytical sensitivity of heterozygote detection in blinded studies was 100%. Snapback genotyping reduced the need for sequencing from 7.9% to 0.5% of PCR products; only 1 amplicon every 5 patients required sequencing to identify nonanticipated rare variants. We identified 2 previously unreported variants: c.3945A>G and c.4243-5C>T. CONCLUSIONS: CFTR analysis by sequential scanning and genotyping with snapback primers is a good match for targeted clinical genetics, for which high analytical accuracy and rapid turnaround times are important.

High-resolution DNA melting analysis in clinical research and diagnostics.

Among nucleic acid analytical methods, high-resolution melting analysis is gaining more and more attention. High-resolution melting provides simple, homogeneous solutions for variant scanning and genotyping, addressing the needs of today's overburdened laboratories with rapid turnaround times and minimal cost. The flexibility of the technique has allowed it to be adopted by a wide range of disciplines for a variety of applications. In this review we examine the broad use of high-resolution melting analysis, including gene scanning, genotyping (including small amplicon, unlabeled probe and snapback primers), sequence matching and methylation analysis. Four major application arenas are examined to demonstrate the methods and approaches commonly used in particular fields. The appropriate usage of high-resolution melting analysis is discussed in the context of known constraints, such as sample quality and quantity, with a particular focus placed on proper experimental design in order to produce successful results.

Spatial DNA melting analysis for genotyping and variant scanning.

A continuous-flow, temperature gradient microfluidic device was used to demonstrate spatial DNA melting analysis with the resolution and reproducibility necessary for clinical SNP scanning and genotyping of human genomic DNA. With a steady-state temperature gradient of 20-30 degrees C across a sample, melting curves were constructed from a single fluorescence data acquisition. This technique was used to scan for heterozygotes and to fully genotype single base changes using unlabeled probes. Signal-to-noise ratios of 150-300 were achieved. The thermal effects of sample flow were examined, and temperature control was aided by inclusion of an isothermal channel inlet and thermal relaxation times in the experimental protocol. Human single base variants examined by spatial DNA melting analysis included rs354439, HTR2A 102T > C, and three alleles that affect appropriate warfarin dosage (CYP2C9*2, CYP2C9*3, and VKORC1 1173C > T). Heterozygote scanning was demonstrated with rs354439, while the other PCR targets were genotyped using unlabeled probes with T(m) differences of approximately 5 degrees C between genotypes. To validate the method, 12 blinded DNA samples were genotyped at the three warfarin-related sites by spatial DNA melting analysis with 100% accuracy.

Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.

BACKGROUND: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. METHODS: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner(R). We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. RESULTS: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. CONCLUSIONS: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.

Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis.

This protocol permits the simultaneous mutation scanning and genotyping of PCR products by high-resolution DNA melting analysis. This is achieved using asymmetric PCR performed in the presence of a saturating fluorescent DNA dye and unlabeled oligonucleotide probes. Fluorescent melting curves of both PCR amplicons and amplicon-probe duplexes are analyzed. The shape of the PCR amplicon melting transition reveals the presence of heterozygotes, whereas specific genotyping is enabled by melting of the unlabeled probe-amplicon duplex. Unbiased hierarchal clustering of melting transitions automatically groups different sequence variants; this allows common variants to be easily recognized and genotyped. This technique may be used in both laboratory research and clinical settings to study single-nucleotide polymorphisms and small insertions and deletions, and to diagnose associated genetic disorders. High-resolution melting analysis accomplishes simultaneous gene scanning and mutation genotyping in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires <30 min (capillaries) or 1.5 h (96- or 384-well plates) and melting acquisition takes 1-2 min per capillary or 5 min per plate.