DNA melting analysis.

Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.

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.

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.

First Report of blaVIM-4- and mcr-9-Coharboring Enterobacter Species Isolated from a Pediatric Patient.

An Enterobacter hormaechei isolate harboring blaVIM-4 and mcr-9 was recovered from a pediatric patient in a U.S. hospital. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9IMPORTANCE We describe the complete genome assembly and sequence of a clinical Enterobacter isolate harboring both blaVIM-4 and mcr-9 recovered from a pediatric patient in the United States with a history of travel to Egypt. Moreover, to the best of our knowledge, this is the first report of an Enterobacter isolate harboring both blaVIM-4 and mcr-9 from the United States. The blaVIM-4 and mcr-9 genes are carried on the same IncH12 plasmid, pME-1a. The isolate tested susceptible to colistin, without observed induction of colistin resistance. The mcr-9 gene is located between two insertion elements, IS903 and IS1, but lacks the downstream regulatory genes (qseC and qseB) found in other isolates that harbor mcr-9.

Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens.

Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexed in vitro diagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus neoformans/Cryptococcus gattii We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, and L. monocytogenes and N. meningitidis were not observed in the study. For S. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

Enhancing pathogen identification in patients with meningitis and a negative Gram stain using the BioFire FilmArray(®) Meningitis/Encephalitis panel.

BACKGROUND: Meningitis with a negative cerebrospinal (CSF) Gram stain represents a diagnostic and therapeutic challenge. The purpose of our study was to evaluate the performance of the BioFire FilmArray(®) Meningitis/Encephalitis (FA ME) panel in patients presenting with community-acquired meningitis with a negative Gram stain. METHODS: CSF from 48 patients with community-acquired meningitis with a negative Gram stain admitted to four hospitals in Houston, TX underwent additional testing by the FA ME. FA ME results were compared to results obtained as part of routine evaluation. RESULTS: The panel detected pathogens not previously identified in 11 (22.9 %) of 48, but did not detect pathogens identified by standard technique (West Nile virus, Histoplasma) in 5 (15.2 %) patients. CONCLUSIONS: Rapid testing for the most common pathogens causing meningitis will aid in the diagnosis and treatment of patients with meningitis.

Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda.

Meningitis remains a worldwide problem, and rapid diagnosis is essential to optimize survival. We evaluated the utility of a multiplex PCR test in differentiating possible etiologies of meningitis. Cerebrospinal fluid (CSF) from 69 HIV-infected Ugandan adults with meningitis was collected at diagnosis (n=51) and among persons with cryptococcal meningitis during therapeutic lumbar punctures (n=68). Cryopreserved CSF specimens were analyzed with BioFire FilmArray® Meningitis/Encephalitis panel, which targets 17 pathogens. The panel detected Cryptococcus in the CSF of patients diagnosed with a first episode of cryptococcal meningitis by fungal culture with 100% sensitivity and specificity and differentiated between fungal relapse and paradoxical immune reconstitution inflammatory syndrome in recurrent episodes. A negative FilmArray result was predictive of CSF sterility on follow-up lumbar punctures for cryptococcal meningitis. EBV was frequently detected in this immunosuppressed population (n=45). Other pathogens detected included: cytomegalovirus (n=2), varicella zoster virus (n=2), human herpes virus 6 (n=1), and Streptococcus pneumoniae (n=1). The FilmArray Meningitis/Encephalitis panel offers a promising platform for rapid meningitis diagnosis.

Immobilization of active human carboxylesterase 1 in biomimetic silica nanoparticles.

The encapsulation of proteins in biomimetic silica has recently been shown to successfully maintain enzymes in their active state. Organophosphate (OP) compounds are used as pesticides as well as potent chemical warfare nerve agents. Because these toxicants are life threatening, we sought to generate biomimetic silicas capable of responding to OPs. Here, we present the silica encapsulation of human drug metabolism enzyme carboxylesterase 1 (hCE1) in the presence of a range of catalysts. hCE1 was successfully encapsulated into silica particles when lysozyme or the peptide R5 were used as catalysts; in contrast, polyethyleneimine, a catalyst used to encapuslate other enzymes, did not facilitate hCE1 entrapment. hCE1 silica particles in a column chromatography format respond to the presence of the OP pesticides paraoxon and dimethyl-p-nitrophenyl phosphate in solution. These results may lead to novel approaches to detect OP pesticides or other weaponized agents that bind hCE1.

Nerve agent hydrolysis activity designed into a human drug metabolism enzyme.

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.

Human carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin.

Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P(R) enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P(S) isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P(S) isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.

Chronic dDAVP infusion in rats decreases the expression of P2Y2 receptor in inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD.

Activation of P2Y2 receptor (P2Y2-R) in inner medullary collecting duct (IMCD) of rat decreases AVP-induced water flow and releases PGE(2). We observed that dehydration of rats decreases the expression of P2Y2 receptor in inner medulla (IM) and P2Y2-R-mediated PGE(2) release by IMCD. Because circulating vasopressin (AVP) levels are increased in dehydrated condition, we examined whether chronic infusion of desmopressin (dDAVP) has a similar effect on the expression and activity of P2Y2-R. Groups of rats were infused with saline or dDAVP (5 or 20 ng/h sc, 5 or 6 days) via osmotic minipumps and euthanized. Urine volume, osmolality, and PGE(2) metabolite content were determined. AQP2- and P2Y2- and V2-R mRNA and/or protein in IM were quantified by real-time RT-PCR and immunoblotting, respectively. P2Y2-R-mediated PGE(2) release by freshly prepared IMCD was assayed using ATPgammaS as a ligand. Chronic dDAVP infusion resulted in low-output of concentrated urine and significantly increased the AQP2 protein abundance in IM. On the contrary, dDAVP infusion at 5 or 20 ng/h significantly decreased P2Y2-R protein abundance (approximately 40% of saline-treated group). In parallel, the relative expression of P2Y2-R vs. AQP2- or V2-R mRNA was significantly decreased. Furthermore, the P2Y2-R-mediated PGE(2) release by IMCD was significantly decreased in rats infused 20 ng/h but not 5 ng/h of dDAVP. Urinary PGE(2) metabolite excretion, however, did not change with dDAVP infusion. In conclusion, chronic dDAVP infusion decreases the expression and activity of P2Y2-R in IM. This may be due to a direct effect of dDAVP or dDAVP-induced increase in medullary tonicity.

P2Y2 receptor-mediated release of prostaglandin E2 by IMCD is altered in hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function.

Circulating vasopressin levels change in hydrated and dehydrated conditions and thus control osmotic water permeability (P(f)) of the inner medullary collecting duct (IMCD). Prostaglandin E2 (PGE2) antagonizes vasopressin-induced P(f) of IMCD. Previously, we showed that activation of P2Y2 receptor (P2Y2-R) in IMCD results in production and release of PGE2, and P2Y2-R mRNA and protein are significantly elevated in inner medullas of hydrated rats compared with dehydrated rats. Therefore, we examined whether the altered expression of P2Y2-R in hydrated and dehydrated states is associated with corresponding changes in P2Y2-R-mediated PGE2 release by the IMCD. Rats were hydrated by providing sucrose water as the sole drinking fluid or dehydrated by water deprivation for 2 days. This resulted in high output-low osmolality and low output-high osmolality urines in hydrated and dehydrated rats, respectively. In hydrated rats, there was a significant increase in tubular fluid PGE2, measured indirectly by assessing the urinary PGE2 metabolite. Stimulation of freshly isolated IMCD preparations in vitro with P2Y2-R agonist (ATPgammaS) showed a marked increase in the release of PGE2 in hydrated rats compared with normal rats. These responses were blunted in the IMCD prepared from dehydrated rats. The P2Y2-R-mediated PGE2 release in the IMCD of hydrated rats was mediated largely by cyclooxygenase (COX)-1 as COX-1-specific inhibitor valeroyl salicylate completely blocked the release. The COX-2-specific inhibitor N5398 had only a modest and insignificant inhibitory effect. In conclusion, the increased sensitivity of purinergic-prostanoid interaction seen in the IMCD of hydrated rats may represent a novel vasopressin-independent regulatory mechanism of IMCD function.