Performance of Fentanyl Immunoassays in an ED Patient Population.

BACKGROUND: Fentanyl is a synthetic opioid fueling the current opioid crisis in the United States. While emergency department (ED) visits due to opioid-related overdoses, injection complications, and withdrawals become increasingly more frequent, fentanyl is not detected in routine toxicology testing. We evaluated 2 FDA-approved fentanyl immunoassays in a sampled ED population. METHODS: De-identified, remnant urine specimens (n = 213) collected from patients presenting to a large ED were analyzed using ARK Fentanyl II (ARK II) and Immunalysis SEFRIA (SEFRIA) fentanyl immunoassays on an Architect c16000 (Abbott) analyzer. All discrepant specimens were evaluated by LC-MS/MS. Additionally, polysubstance abuse patterns and trends were analyzed. RESULTS: While intra-assay imprecision was comparable for ARK II and SEFRIA, inter-assay imprecision for ARK II and SEFRIA varied from 8.0% to 1.8% and from 37% to 12.5%, respectively. SEFRIA had a marginally higher false-positivity rate (3%) than ARK II (1%). Both assays had equivalent sensitivity of 95%, with ARK II (99%) having greater specificity than SEFRIA (97%). Fentanyl was detected in 13.7% of drug-panel-positive patient samples and most frequently observed in patients also testing positive for amphetamines and cocaine. Notably, fentanyl was detected in 5.3% of patient samples that were negative for all other drugs in our standard toxicology panel. CONCLUSIONS: A sizable portion of drug-positive samples from our ED were positive for fentanyl, with a subset of patients testing positive for fentanyl alone. Implementation of fentanyl testing into routine toxicology panels can elucidate polysubstance abuse paradigms and capture ED patients that would go undetected in standard panels.

Implications of Below-Ground Allelopathic Interactions of Camelina sativa and Microorganisms for Phosphate Availability and Habitat Maintenance.

Toxic breakdown products of young Camelina sativa (L.) Crantz, glucosinolates can eliminate microorganisms in the soil. Since microorganisms are essential for phosphate cycling, only insensitive microorganisms with phosphate-solubilizing activity can improve C. sativa's phosphate supply. In this study, 33P-labeled phosphate, inductively coupled plasma mass spectrometry and pot experiments unveiled that not only Trichoderma viride and Pseudomonas laurentiana used as phosphate-solubilizing inoculants, but also intrinsic soil microorganisms, including Penicillium aurantiogriseum, and the assemblies of root-colonizing microorganisms solubilized as well phosphate from apatite, trigger off competitive behavior between the organisms. Driving factors in the competitiveness are plant and microbial secondary metabolites, while glucosinolates of Camelina and their breakdown products are regarded as key compounds that inhibit the pathogen P. aurantiogriseum, but also seem to impede root colonization of T. viride. On the other hand, fungal diketopiperazine combined with glucosinolates is fatal to Camelina. The results may contribute to explain the contradictory effects of phosphate-solubilizing microorganisms when used as biofertilizers. Further studies will elucidate impacts of released secondary metabolites on coexisting microorganisms and plants under different environmental conditions.

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products.

Persistent Elevation of Troponin I in a Pediatric Patient Resulting From Macrotroponin Complex.

ABSTRACT: Although chest pain is a common chief complaint among pediatric patients, cardiac pathology historically has accounted for a small percentage of cases. However, the emergence of COVID-19 and particularly its potential for leading to multisystem inflammatory syndrome has changed the threshold for the evaluation of cardiac etiologies of chest pain. This evaluation often includes measurement of the serum cardiac troponin I level. We present a case of a 16-year-old male athlete who presented to an outside emergency department with chest pain and was found to have elevated serum troponin I levels. Despite sports restriction, his troponin level remained elevated for months in the absence of other clinical findings and he was subsequently referred to our outpatient pediatric cardiology clinic. Further laboratory evaluation revealed that, in addition to troponin I, the assay measured an immune complex of uncertain significance formed by anti-troponin I antibodies bound to troponin I, known as macrotroponin. Delayed clearance of this complex from the bloodstream can result in overestimation of troponin I levels that can affect clinical management and create anxiety for our patients and their families. Macrotroponin complex deserves increased recognition among the research and clinical communities, especially in the pediatric realm.

Triiodothyroacetic Acid Cross-Reacts With Measurement of Triiodothyronine (T3) on Various Immunoassay Platforms.

OBJECTIVES: Thyroid hormone analog 3,5,3'-triiodothyroacetic acid (TRIAC) is effective in reducing the hypermetabolism in monocarboxylate transporter 8 (MCT8)-deficient individuals. Because of the structural similarity between TRIAC and 3,3',5'-triiodothyronine (T3), we sought to investigate the degree of cross-reactivity of TRIAC with various commercially available total and free T3 assays. METHODS: Varying concentrations (50-1,000 ng/dL) of TRIAC (Sigma Aldrich) were added to pooled serum and assayed for total T3 (TT3) and free T3 (FT3) on the following platforms: e602 (Roche Diagnostics), Architect (Abbott Diagnostics), Centaur (Siemens Healthcare Diagnostics), IMMULITE (Siemens Healthcare Diagnostics), DxI (Beckman Coulter), and Vitros (Ortho Clinical Diagnostics). TT3 competition assay with TRIAC was performed by adding increasing amounts of T3 to pooled serum samples that contained a constant concentration of TRIAC (250 ng/dL). RESULTS: Significant overestimation of TT3 and FT3 assays were observed across all platforms corresponding to increasing concentrations of TRIAC. The TRIAC effect at 250 ng/dL showed a constant interference of approximately 190 ng/dL TT3. CONCLUSIONS: All commercial TT3 and FT3 assays tested in this work cross-react significantly with TRIAC. Therefore, patients undergoing TRIAC therapy should have T3 hormone response monitored using alternative nonimmunoassay-based methods to avoid misinterpretation of thyroid function profiles.

Predicting drug targets by homology modelling of Pseudomonas aeruginosa proteins of unknown function.

The efficacy of antibiotics to treat bacterial infections declines rapidly due to antibiotic resistance. This problem has stimulated the development of novel antibiotics, but most attempts have failed. Consequently, the idea of mining uncharacterized genes of pathogens to identify potential targets for entirely new classes of antibiotics was proposed. Without knowing the biochemical function of a protein, it is difficult to validate its potential for drug targeting; therefore, the functional characterization of bacterial proteins of unknown function must be accelerated. Here, we present a paradigm for comprehensively predicting the biochemical functions of a large set of proteins encoded by hypothetical genes in human pathogens to identify candidate drug targets. A high-throughput approach based on homology modelling with ten templates per target protein was applied to the set of 2103 P. aeruginosa proteins encoded by hypothetical genes. The >21000 homology modelling results obtained and available biological and biochemical information about several thousand templates were scrutinized to predict the function of reliably modelled proteins of unknown function. This approach resulted in assigning one or often multiple putative functions to hundreds of enzymes, ligand-binding proteins and transporters. New biochemical functions were predicted for 41 proteins whose essential or virulence-related roles in P. aeruginosa were already experimentally demonstrated. Eleven of them were shortlisted as promising drug targets that participate in essential pathways (maintaining genome and cell wall integrity), virulence-related processes (adhesion, cell motility, host recognition) or antibiotic resistance, which are general drug targets. These proteins are conserved in other WHO priority pathogens but not in humans; therefore, they represent high-potential targets for preclinical studies. These and many more biochemical functions assigned to uncharacterized proteins of P. aeruginosa, made available as PaPUF database, may guide the design of experimental screening of inhibitors, which is a crucial step towards the validation of the highest-potential targets for the development of novel drugs against P. aeruginosa and other high-priority pathogens.

AACC Practical Recommendations for Implementing and Interpreting SARS-CoV-2 Emergency Use Authorization and Laboratory-Developed Test Serologic Testing in Clinical Laboratories.

BACKGROUND: The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous US Food and Drug Administration emergency use authorization (EUA) and laboratory-developed test (LDT) serologic assays have become available. The performance characteristics of these assays and their clinical utility continue to be defined in real time during this pandemic. The AACC convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories; the in vitro diagnostics industry; and regulatory agencies to provide practical recommendations for implementation and interpretation of these serologic tests in clinical laboratories. CONTENT: The currently available EUA serologic tests and platforms, information on assay design, antibody classes including neutralizing antibodies, and the humoral immune responses to SARS-CoV-2 are discussed. Verification and validation of EUA and LDT assays are described, along with a quality management approach. Four indications for serologic testing are outlined. Recommendations for result interpretation, reporting comments, and the role of orthogonal testing are also presented. SUMMARY: This document aims to provide a comprehensive reference for laboratory professionals and healthcare workers to appropriately implement SARS-CoV-2 serologic assays in the clinical laboratory and to interpret test results during this pandemic. Given the more frequent occurrence of outbreaks associated with either vector-borne or respiratory pathogens, this document will be a useful resource in planning for similar scenarios in the future.

Comparative analysis of antibodies to SARS-CoV-2 between asymptomatic and convalescent patients.

The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral Spike protein, and approximately 3% were positive. Of these 3%, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them with those of asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Furthermore, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or post vaccination.

Cefoxitin-Serum Creatinine Interference in a Patient With Nontuberculous Mycobacteria Ventriculomeningitis.

Cefoxitin is a second-generation cephamycin antibiotic, which at concentrations ≥100 µg/mL is known to modestly interfere, for up to 2 hours post-infusion, with serum creatinine measurement via the traditional Jaffe-based assay. We report a case of a severe serum creatinine elevation while utilizing cefoxitin as a component of an antimicrobial regimen in a critically ill patient with Mycobacterium abscessus ventriculomeningitis. Our results, both via patient serum analysis and a cefoxitin spiking experiment, demonstrate interference despite the utilization of improved modern Jaffe-based assays. In fact, the cefoxitin-creatinine interference may be clinically relevant at concentrations 3 times lower than that listed in the package insert and may display more than a modest interference at typical therapeutic concentrations.

The Effect of Sample Handling on Free Valproic Acid Levels.

BACKGROUND: Valproic acid (VPA) is a broad-spectrum anticonvulsant drug. Under normal conditions, this drug is highly protein bound. However, in patients with hypoalbuminemia, the free fraction can increase substantially while the total VPA levels remain in therapeutic range. The neurologic activity and toxicity of the drug are directly related to free drug levels. METHODS: Our in-house free VPA assay was validated using 20 patient samples obtained from a reference laboratory (RL1). It was further evaluated by parallel testing with RL1 using samples collected from our patients. Subsequently, sample handling effects were investigated by comparing free VPA levels measured in our laboratory to 3 selected RLs with different sample transportation conditions. RESULTS: No significant bias was observed between the in-house assay (y) and RL1 (x) assay in free VPA measurement (y = 1.12x + 0.072, r = 0.994). However, patient samples collected in our institution and sent to RL1 revealed significant negative bias (y = 0.776x - 3.861, r = 0.954). A large discrepancy in free VPA levels was further observed from identical aliquots of the same samples transported to 3 RLs in different conditions. CONCLUSIONS: Our study demonstrated that sample handling has significant impact on free VPA levels. The observed magnitude of variation exceeds a clinically acceptable limit and could alter clinical decisions.

Evaluation of Orthogonal Testing Algorithm for Detection of SARS-CoV-2 IgG Antibodies.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody testing is an important tool in assessment of pandemic progress, contact tracing, and identification of recovered coronavirus disease 2019 (COVID-19) patients. We evaluated an orthogonal testing algorithm (OTA) to improve test specificity in these use cases. METHODS: A two-step OTA was applied where individuals who initially tested positive were tested with a second test. The first-line test, detecting IgG antibodies to the viral nucleocapsid protein, was validated in 130 samples and the second-line test, detecting IgG antibodies to the viral spike protein in 148 samples. The OTA was evaluated in 4333 clinical patient specimens. The seropositivity rates relative to the SARS-CoV-2 PCR positivity rates were evaluated from our entire patient population data (n = 5102). RESULTS: The first-line test resulted in a clinical sensitivity of 96.4% (95% CI; 82.3% to 99.4%), and specificity of 99.0% (95% CI; 94.7% to 99.8%), whereas the second-line test had a sensitivity of 100% (95% CI; 87.1% to 100%) and specificity of 98.4% (95% CI; 94.2% to 99.5%). Using the OTA, 78/98 (80%) of initially positive SARS-CoV-2 IgG results were confirmed with a second-line test, while 11/42 (26%) of previously diagnosed COVID-19 patients had no detectable antibodies as long as 94 days post PCR diagnosis. CONCLUSION: Our results show that an OTA can be used to identify patients who require further follow-up due to potential SARS CoV-2 IgG false positive results. In addition, serological testing may not be sufficiently sensitive to reliably detect prior COVID-19 infection.

Center-related Bias in MELD Scores Within a Liver Transplant UNOS Region: A Call for Standardization.

BACKGROUND: Model for End-Stage Liver Disease (MELD) score-based liver transplant allocation was implemented as a fair and objective measure to prioritize patients based upon disease severity. Accuracy and reproducibility of MELD is an essential assumption to ensure fairness in organ access. We hypothesized that variability in laboratory methodology between centers could impact allocation scores for individuals on the transplant waiting list. METHODS: Aliquots of 30 patient serum samples were analyzed for creatinine, bilirubin, and sodium in all transplant centers within United Network for Organ Sharing (UNOS) region 9. Descriptive statistics, intraclass correlation coefficients (ICCs), and linear mixed-effects regression were used to determine the relationship between center, bilirubin, and calculated MELD-sodium (MELD-Na) score. RESULTS: The mean MELD-Na score per sample ranged from 14 to 38. The mean range in MELD-Na per sample was 3 points, but 30% of samples had a range of 4-6 points. Correlation plots and intraclass correlation coefficient analysis confirmed bilirubin interfered with creatinine, with worsening agreement in creatinine at high bilirubin levels. Center and bilirubin were independently associated with creatinine reported in mixed-effects models. Unbiased hierarchical clustering suggested that samples from specific centers have consistently higher creatinine and MELD-Na values. CONCLUSIONS: Despite implementation of creatinine standardization, centers within a single UNOS region report clinically significant differences in MELD-Na on an identical sample, with differences of up to 6 points in high MELD-Na patients. The bias in MELD-Na scores based upon center choice within a region should be addressed in the current efforts to eliminate disparities in liver transplant access.

Comparison of the Abbott Architect BRAHMS and the Biomérieux Vidas BRAHMS Procalcitonin Assays.

BACKGROUND: Procalcitonin (PCT) is a well-established marker for bacterial infection. Recently the US Food and Drug Administration approved the expanded use of this biomarker to guide clinical decisions for antibiotic treatment in patients with lower respiratory tract infections. Both the Architect BRAHMS PCT (PCT-A) and Vidas BRAHMS PCT (PCT-V) are approved for this indication. The aim of this study is to evaluate analytical performance of PCT-A in comparison to PCT-V. METHODS: PCT-A and PCT-V were evaluated for intra- and interassay precision and functional sensitivity. To assess the accuracy of PCT-A, 108 residual plasma specimens were randomly selected from routine hospital orders, and PCT was measured concurrently with PCT-A and PCT-V. RESULTS: Both assays demonstrated excellent precision, with intraassay precision ranging from 2.2% to 4.0% CV and interassay precision ranging from 2.5% to 3.6% CV. The functional sensitivity was verified at 0.01 ng/mL for PCT-A and at 0.05 ng/mL for PCT-V. The Passing-Bablok regression revealed approximately 20% negative bias of PCT-A compared to PCT-V (PCT-A = 0.042 + 0.79 × PCT-V, r = 0.995). The concordance of the 2 methods at diagnostically important cutoffs (0.10, 0.25, 0.50, and 2.0 ng/mL) was excellent, with overall agreement >93% at each threshold. CONCLUSION: The results of our study show improved sensitivity and equivalent clinical performance of PCT-A compared to PCT-V. The availability of this test on common clinical immunoassay analyzers may help accelerate its adoption into antimicrobial stewardship programs and thereby improve antibiotic use and patient outcomes.

Monodisperse Sequence-Controlled α-l-Fucosylated Glycooligomers and Their Multivalent Inhibitory Effects on LecB.

The opportunistic bacterium Pseudomonas aeruginosa, often exhibiting multiresistance against conventional antibiotics, expresses the lectin LecB that is suspected to be an important factor during biofilm formation via interactions with cell-surface presented carbohydrate ligands such as the blood group antigens. Therefore, carbohydrate-based ligands interfering with LecB binding have the potential to lead to new anti-biofilm and anti-adhesion therapies. This study explores in vitro binding potencies of glycomimetic ligands containing up to six α-l-fucose ligands on a monodisperse, sequence-controlled oligoamide scaffold interacting with LecB. Surface plasmon resonance (SPR) and a modified enzyme-linked lectin assay (mELLA) revealed an increasing affinity to LecB with increasing fucose valency. Furthermore, fucosylated glycooligomers were shown to inhibit the formation of P. aeruginosa biofilm up to 20%. Overall these results show the potential of fucosylated oligoamides to be further developed as inhibitors of LecB binding and biofilm formation.

Quality Control Practices for Chemistry and Immunochemistry in a Cohort of 21 Large Academic Medical Centers.

OBJECTIVES: In the United States, minimum standards for quality control (QC) are specified in federal law under the Clinical Laboratory Improvement Amendment and its revisions. Beyond meeting this required standard, laboratories have flexibility to determine their overall QC program. METHODS: We surveyed chemistry and immunochemistry QC procedures at 21 clinical laboratories within leading academic medical centers to assess if standardized QC practices exist for chemistry and immunochemistry testing. RESULTS: We observed significant variation and unexpected similarities in practice across laboratories, including QC frequency, cutoffs, number of levels analyzed, and other features. CONCLUSIONS: This variation in practice indicates an opportunity exists to establish an evidence-based approach to QC that can be generalized across institutions.

Evaluation of direct-to-consumer low-volume lab tests in healthy adults.

BACKGROUND: Clinical laboratory tests are now being prescribed and made directly available to consumers through retail outlets in the USA. Concerns with these test have been raised regarding the uncertainty of testing methods used in these venues and a lack of open, scientific validation of the technical accuracy and clinical equivalency of results obtained through these services. METHODS: We conducted a cohort study of 60 healthy adults to compare the uncertainty and accuracy in 22 common clinical lab tests between one company offering blood tests obtained from finger prick (Theranos) and 2 major clinical testing services that require standard venipuncture draws (Quest and LabCorp). Samples were collected in Phoenix, Arizona, at an ambulatory clinic and at retail outlets with point-of-care services. RESULTS: Theranos flagged tests outside their normal range 1.6× more often than other testing services (P < 0.0001). Of the 22 lab measurements evaluated, 15 (68%) showed significant interservice variability (P < 0.002). We found nonequivalent lipid panel test results between Theranos and other clinical services. Variability in testing services, sample collection times, and subjects markedly influenced lab results. CONCLUSION: While laboratory practice standards exist to control this variability, the disparities between testing services we observed could potentially alter clinical interpretation and health care utilization. Greater transparency and evaluation of testing technologies would increase their utility in personalized health management. FUNDING: This work was supported by the Icahn Institute for Genomics and Multiscale Biology, a gift from the Harris Family Charitable Foundation (to J.T. Dudley), and grants from the NIH (R01 DK098242 and U54 CA189201, to J.T. Dudley, and R01 AG046170 and U01 AI111598, to E.E. Schadt).

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation-emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.

The Mount Sinai clinical pathway for the management of pheochromocytoma.

OBJECTIVE: Pheochromocytomas are complex tumors that require a comprehensive and systematic management plan orchestrated by a multidisciplinary team. METHODS: To achieve these ends, The Mount Sinai Adrenal Center hosted an interdisciplinary retreat where experts in adrenal disorders assembled with the aim of developing a clinical pathway for the management of pheochromocytomas. RESULTS: The result was a consensus for the diagnosis, perioperative management, and postoperative management of pheochromocytomas, with specific recommendations from our team of adrenal experts, as well as a review of the current literature. CONCLUSION: Our clinical pathway can be applied by other institutions directly or may serve as a guide for institution-specific management.