Advances in TB testing.

Globally, tuberculosis (TB) was the leading cause of death from a single infectious agent until the coronavirus (COVID-19) pandemic. In 2020, an estimated 10 million people fell ill with TB and a total of 1.5 million people died from the disease. About one-quarter of the global population, almost two billion people, is estimated to be latently infected with Mycobacterium tuberculosis (MTB). Although latent TB infection (LTBI) is asymptomatic and noncontagious, about 5-10% of LTBI patients have a lifetime risk of progression to active TB. The diagnosis and treatment of active cases are extremely vital for TB control programs. However, achieving the End TB goal of 2035 without the ability to identify and treat the pool of latently infected individuals will be a big challenge. To do so, improved technology to provide more accurate diagnostic tools and accessibility are crucial. Therefore, this chapter covers the current WHO-endorsed tests and advances in diagnostic and screening tests for active and latent TB.

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity.

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3-97.2%) compared to 91.7% (95% CI 77.5-98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3-100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

Missed critical value callbacks due to middleware flaw.

OBJECTIVES: In 2018, our clinical laboratory was alerted to back-to-back plasma sodium critical value callback failures on the same patient, occurring on different shifts and involving different technologists. Therefore, we set forth to investigate the root cause for the critical value callback failures. DESIGN AND METHODS: We conducted a thorough investigation focused on the processes associated with critical value identification and notification for plasma sodium measurement performed on the Siemens Vista. RESULTS: Our investigation uncovered a flaw in the Siemens CentraLink middleware software. A default dark blue bar in the top row of the results review display was determined to obscure the red color which highlights critical values for lab staff identification. Sodium was disproportionately impacted by this flaw, as it is commonly ordered as part of metabolic panels, and is listed first among the panel analytes in the top row of the CentraLink display. Retrospective data review comparing critical callback failure rates for sodium to potassium and hemoglobin confirmed that sodium had significantly higher critical callback failure rates than these other analytes. After alerting the product manufacturer, Siemens programmed the CentraLink display so that the top row was blank and devoid of patient results, so that the blue color in the top row would no longer obscure the red visual cue of a patient's critical result. Sodium critical value callback failures were reduced to 0% after this middleware display correction. CONCLUSIONS: Middleware design flaws can have unexpected consequences on clinical laboratory operations. We encourage clinical laboratories to closely examine user interfaces utilized by laboratory staff, and be wary of potential impacts that the display format may have on results reporting.

Performance of an Advanced Interferon-Gamma Release Assay for Mycobacterium tuberculosis Detection.

BACKGROUND: The LIAISON® QuantiFERON®-TB Gold Plus (QFT-Plus) assay, a fully automated chemiluminescence immunoassay (CLIA) system, has recently received FDA approval for the detection of interferon-γ (IFN-γ) on the LIAISON XL analyzer. Here, we evaluated the diagnostic performance of the LIAISON's CLIA method in comparison to the widely used ELISA method using the Qiagen QuantiFERON-TB Gold Plus Blood Collection Tubes. METHODS: Heparinized blood samples from 329 participants were categorized into 3 cohorts, including 15 with confirmed tuberculosis (TB) (active TB cohort), 129 non-TB (low-risk cohort), and 185 potential TB (mixed risk cohort). Samples were analyzed with both assays and results were interpreted according to the manufacturers' criteria. RESULTS: The LIAISON CLIA assay showed an overall agreement with the Qiagen ELISA assay in 13/14 (92.8%) samples among the active cohort, 93/95 (97.9%) among the low-risk cohort, with a Cohen's kappa value of 0.76 and 0.74, respectively. Test results for 185 mixed risk cohort participants showed 97.8% agreement between the LIAISON and the Qiagen, with 17 positive, 163 negative, and 1 indeterminate. Four samples were discrepant; 3 of these were negative on the ELISA but positive on the CLIA assay. CONCLUSION: Overall, the results were comparable (>92% agreement) in our study cohorts. In addition, our mixed risk cohort results showed an excellent agreement of 0.88 (Cohen's kappa value) between the 2 assays. These findings suggest that the automated LIAISON QFT-Plus assay has a comparable diagnostic performance to the Qiagen assay and can be used for latent TB infection (LTBI) diagnosis.

Transient hyperphosphatasemia following pediatric liver transplantation in a patient with hepatic and skeletal abnormalities.

BACKGROUND: We report a unique case of transient hyperphosphatasemia in a pediatric patient with a history of hepatic and skeletal abnormalities. PATIENT AND METHODS: A 2-month old male was diagnosed with progressive familial intrahepatic cholestasis type-2 and osteoporosis after marked increases in liver function tests were noted at 1 month of age. He underwent a second liver transplantation at 1 y. The increased liver function test trend resolved a few weeks post-transplantation. Four months after successful liver transplantation, unexplained significant increases in alkaline phosphatase (ALP) were observed, and they persisted for almost 9 months. Among the etiologies under consideration for the isolated increased ALP activity were viral infections and macro-ALP. RESULTS: A persistent trend in abnormally increased ALP for 9 months was investigated leading to a confirmed diagnosis of transient hyperphosphatasemia (TH). CONCLUSION: Pediatric post-liver transplant patients with skeletal and hepatic abnormalities including isolated markedly increased ALP activities represent a previously undescribed TH patient population. The 4.3% prevalence of TH in pediatric liver transplant recipients within our healthcare system is considerably higher than the previously reported prevalence of 2.1% for patients within the United States.

Heparin-mediated antibiotic delivery from an electrochemically-aligned collagen sheet.

BACKGROUND: Implantable medical devices and hardware are prolific in medicine, but hardware associated infections remain a major issue. OBJECTIVE: To develop and evaluate a novel, biologic antimicrobial coating for medical implants. METHODS: Electrochemically compacted collagen sheets with and without crosslinked heparin were synthesized per a protocol developed by our group. Sheets were incubated in antibiotic solution (gentamicin or moxifloxacin) overnight, and in vitro activity was assessed with five-day diffusion assays against Pseudomonas aeruginosa. Antibiotic release over time from gentamicin-infused sheets was determined using in vitro elution and high performance liquid chromatography (HPLC). RESULTS: Collagen-heparin-antibiotic sheets demonstrated larger growth inhibition zones against P. aeruginosa compared to collagen-antibiotic alone sheets. This activity persisted for five days and was not impacted by rinsing sheets prior to evaluation. Rinsed collagen-antibiotic sheets did not produce any inhibition zones. Elution of gentamicin from collagen-heparin-gentamicin sheets was gradual and remained above the minimal inhibitory concentration for gentamicin-sensitive organisms for 29 days. Conversely, collagen-gentamicin sheets eluted their antibiotic load within 24 hours. Overall, heparin-associated sheets demonstrated larger inhibition zones against P. aeruginosa and prolonged elution profile via HPLC. CONCLUSION: We developed a novel, local antibiotic delivery system that could be used to coat medical implants/hardware in the future and reduce post-operative infections.

Association of RETN +299(G>A) polymorphism with type two diabetes mellitus.

The global prevalence of type-two diabetes mellitus (T2DM) makes it a disease of public health concern. T2DM is strongly linked with insulin resistance caused by increased levels of visceral fat. Visceral fat secretes several adipocytokines that regulate body metabolism. Resistin is one of these adipocytokines which is encoded by the RETN gene. Herein, we tested the association of the RETN +299(G>A) and -420(C>G) single nucleotide polymorphisms (SNPs) with T2DM. T2DM patients (n=282) and healthy subjects (n=125) were included in the study. Subjects with metabolic syndromes other than diabetes were excluded. Genotyping of subjects was performed using PCR-RFLP. The +299(G>A) SNP was associated with T2DM (P=0.038). The AA genotype was higher in T2DM (17%) compared to controls (8%) with an odd ratio of 2.16 and 95% CI of 1.34 to 4.56. With respect to -420(C>G) SNP, no significant association was found with the risk of T2DM (P=0.128). The haplotype analysis of the examined SNPs indicated that the CA haplotype of the -420 and +299 SNPs in RETN was associated with T2DM risk (P=0.004; odd ration 4.0, 95% CI: 1.56-10.0). In conclusion, the present findings suggest a role of the RETN locus in modulating the risk of T2DM.

Initial determination of COVID-19 seroprevalence among outpatients and healthcare workers in Minnesota using a novel SARS-CoV-2 total antibody ELISA.

OBJECTIVES: To avoid the significant risks posed by the use of COVID-19 serology tests with supply chain constraints or poor performance characteristics, we developed an in-house SARS-CoV-2 total antibody test. Our test was compared with three commercial methods, and was used to determine COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. METHODS: Seventy-nine plasma and serum samples from 50 patients 4-69 days after symptom onset who tested positive by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab were used to evaluate our test's clinical performance. Seropositive samples were analyzed for IgG titers in a follow-up assay. Thirty plasma and serum from 12 patients who tested negative by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab and 210 negative pre-pandemic serum samples were also analyzed. Among samples from patients > 14 days after symptom onset, the assay had 100% clinical sensitivity and 100% clinical specificity, 100% positive predictive value and 100% negative predictive value. Analytical specificity was 99.8%, indicating minimal cross-reactivity. A screening study was conducted to ascertain COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. RESULTS: Analysis of serum collected between April 13 and May 21, 2020 indicated a COVID-19 seroprevalence of 2.96% among 1,282 healthcare workers and 4.46% among 2,379 outpatients. CONCLUSIONS: Our in-house SARS-CoV-2 total antibody test can be used to conduct reliable epidemiological studies to inform public health decisions during the COVID-19 pandemic.

Evaluation of an Ion-Exchange HPLC Device for HbA1c Measurement.

BACKGROUND: The ADAMS™ HA-8180V is the 8th generation of a fully automated ion-exchange HPLC system from ARKRAY, and the first to be released onto the US market. We evaluated the HA-8180V, for routine hemoglobin A1c measurement in comparison with the Roche Cobas c501, the Tosoh G8 analyzer for normal hemoglobin, and with the Trinity analyzer for hemoglobin variants. METHODS: The analytical performance (linearity, precision, carryover, and sample stability) was assessed based on the Clinical and Laboratory Standards Institute (CLSI) and manufacturer guidelines. A comparison of the HA-8180V against two major analytical methods was performed for 100 whole blood samples. HA-8180V variant mode was also compared against the Trinity ultra2 A1c analyzer for 50 samples containing hemoglobin variants (HbC 14, HbS 14, HbD 12, and HbE 10). RESULTS: The within-run and total CVs were <0.01 and 0.75% at low HbA1c concentration and 0.46 and 0.63% at high HbA1c concentration, respectively. Linearity was shown in the concentration range 3.4-18.1% HbA1c, carryover was 0.00%, and stability values were excellent. Method comparison demonstrated a high concordance between methods. CONCLUSION: The eighth generation ADAMS HA-8180V A1c analyzer demonstrated high analytical performance adequate for routine clinical use.

Evaluation of automatic mixing versus manual mixing for point of care hemoglobin measurement.

OBJECTIVE: We compared hemoglobin results from manually mixed blood gas (BG) Portex syringes (Smiths Medical) and automatically mixed safePICO BG syringes (Radiometer Medical) measured on the ABL90 FLEX blood gas analyzer at the Point of Care with a laboratory hematology analyzer (XN-series, Sysmex) to determine whether automatic mixing improved the accuracy and precision of hemoglobin measurement relative to the standardized method. METHODS: Ninety-nine lithium heparin tubes and EDTA tubes were collected simultaneously from selected patients at five participating institutions. The lithium heparin tubes were then split between the Portex ABG syringe and the safePICO aspirator. The Portex syringe was mixed manually according to routine procedures, while the safePICO syringe was mixed automatically on the ABL90 FLEX using the automatic mixing ball. The two syringes were mixed and run on the ABL90 FLEX analyzer by the same lab personnel. Total hemoglobin results obtained from the EDTA tubes on the Sysmex XN hematology analyzer were used as the reference values. RESULTS: Manual mixing demonstrated a larger scatter of hemoglobin values (R2 â€‹= â€‹0.515 and Mean diff.: -0.9 â€‹g/dL) while automatic mixing at the point of care yielded a significantly better correlation (R2 â€‹= â€‹0.986 and Mean diff.: -0.2 â€‹g/dL) when compared to a reference hematology analyzer. CONCLUSION: Using the safePICO syringe with automatic mixing at the point of care results in a significantly better correlation of hemoglobin with the standardized method.

S-Nitrosylation of CHIP Enhances F508Del-CFTR Maturation.

S-nitrosothiols (SNOs) are endogenous signaling molecules that have numerous beneficial effects on the airway via cyclic guanosine monophosphate-dependent and -independent processes. Healthy human airways contain SNOs, but SNO levels are lower in the airways of patients with cystic fibrosis (CF). In this study, we examined the interaction between SNOs and the molecular cochaperone C-terminus Hsc70 interacting protein (CHIP), which is an E3 ubiquitin ligase that targets improperly folded CF transmembrane conductance regulator (CFTR) for subsequent degradation. Both CFBE41o- cells expressing either wild-type or F508del-CFTR and primary human bronchial epithelial cells express CHIP. Confocal microscopy and IP studies showed the cellular colocalization of CFTR and CHIP, and showed that S-nitrosoglutathione inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the mature and immature forms of F508del-CFTR. In fact, SNO inhibition of the function and expression of CHIP not only improved the maturation of CFTR but also increased CFTR's stability at the cell membrane. S-nitrosoglutathione-treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than cells that were not treated, suggesting that the S-nitrosylation of CHIP prevents the ubiquitination of CFTR by inhibiting CHIP's E3 ubiquitin ligase function. Furthermore, the exogenous SNOs S-nitrosoglutathione diethyl ester and S-nitro-N-acetylcysteine increased the expression of CFTR at the cell surface. After CHIP knockdown with siRNA duplexes specific for CHIP, F508del-CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del-CFTR expression on airway epithelial cell surfaces. Together, these findings indicate that S-nitrosylation of CHIP is a novel mechanism of CFTR correction, and we anticipate that these insights will allow different SNOs to be optimized as agents for CF therapy.

Cyclic compression increases F508 Del CFTR expression in ciliated human airway epithelium.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

Pharmacokinetic study of Sudaxine in dog plasma using novel LC-MS/MS method.

CONTEXT: Sudaxine is a novel respiratory stimulant that increases ventilatory drive via NO+ -thiolate signaling and is under development for reversal of opioid-induced respiratory depression and other critical care indications. OBJECTIVE: This study investigates the pharmacokinetic characteristics after intravenous administration of Sudaxine by using a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. MATERIALS AND METHODS: A sensitive LC-MS/MS method was validated to determine the concentration of Sudaxine in beagle dog plasma after intravenous administration of Sudaxine at (3, 10, 30, and 100 mg/kg). Blood samples (1 mL) were collected at designated time points and SDX concentration was measured for pharmacokinetic study. RESULTS: The calibration curve was linear within the range of 50-5,000 ng/mL with the lower limit of quantification at 50 ng/mL. The CTmax for all doses was reached at 10 minutes (Tmax ). Over the dose range studied, average concentration - time curves and systemic exposure (CTmax and AUC0-t ) increased to Sudaxine dose. The terminal half-life of Sudaxine in dogs ranged from 10 to 30 minutes and about 17.3 ± 1.0% of Sudaxine was protein-bound in dog plasma. DISCUSSION AND CONCLUSIONS: We developed a novel LC-MS/MS method of Sudaxine detection and quantification and determined its pharmacokinetic profiles after intravenous administration in canine subjects. Sudaxine followed first-order kinetics with rapid dose-dependent clearance rates and short half-life making it an ideal candidate for use in a critical care setting with intramuscular or IV administration.

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Endothelial nitric oxide synthase oxygenase on lipid nanodiscs: A nano-assembly reflecting native-like function of eNOS.

Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∼65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain.

cAMP-dependent activation of protein kinase A attenuates respiratory syncytial virus-induced human airway epithelial barrier disruption.

Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC proteins by confocal microscopy. Increased intracellular cAMP level significantly attenuated RSV-induced disassembly of AJC. These barrier-protective effects of cAMP were due to the activation of PKA signaling and did not involve Epac activity. Increased cAMP level reduced RSV-induced reorganization of the actin cytoskeleton, including apical accumulation of an essential actin-binding protein, cortactin, and inhibited expression of the RSV F protein. These barrier-protective and antiviral-function of cAMP signaling were evident even when cAMP level was increased after the onset of RSV infection. Taken together, our study demonstrates that cAMP/PKA signaling attenuated RSV-induced disruption of structure and functions of the model airway epithelial barrier by mechanisms involving the stabilization of epithelial junctions and inhibition of viral biogenesis. Improving our understanding of the mechanisms involved in RSV-induced epithelial dysfunction and viral pathogenesis will help to develop novel anti-viral therapeutic approaches.